Composition for cryopreservation of cells, method for freezing cells, and cell preparation

Abstract

Provided is a composition for cryopreservation of cells, which is capable of protecting cells at the time of freezing of the cells, for example, even if substantially no organic solvent-based cell cryoprotectant is added. The composition for cryopreservation of cells of the present invention contains a sugar or a derivative thereof and / or a surfactant.

Description

Technical Field

[0001] This invention relates to a composition for cell cryopreservation, a method for freezing cells, and cell preparations. Background Technology

[0002] With the development of regenerative medicine, cells isolated from organisms are cultured in large quantities and further processed, such as through gene delivery. Furthermore, there are attempts to use these cultured or processed cells as cell-based drugs for disease treatment.

[0003] In the manufacturing process of the cell-based drug, after the cells are cultured and processed in a cell processing facility, the processed cells need to be cryopreserved in order to transport them to hospitals (Non-Patent Documents 1 and 2). In this cryopreservation, the processed cells are dispersed in a cell cryopreservation solution containing a cell cryoprotectant and then frozen. Subsequently, they are transported in a frozen cell state.

[0004] Existing technical documents Non-patent literature Non-patent literature 1: Wang, J., Li, R., "Effects, methods and limits of thecryopreservation on mesenchymal stem cells.", Stem Cell Res Ther, 2024, 15,Article number: 337 Non-patent literature 2: Murray KA, Gibson MI., “Chemical approaches tocryopreservation.”, Nat Rev Chem., 2022, 6(8), p.579–593. Summary of the Invention

[0005] The problem that the invention aims to solve As a cryopreservation solution, dimethyl sulfoxide (DMSO)-containing cryopreservation solutions are widely used as cell cryoprotectants due to their high cell cryoprotective effect. However, because DMSO is cytotoxic, it has been reported that DMSO-containing cell drugs... in vivo (In vivo) administration can exhibit toxicity. In addition to DMSO, organic solvent-based cell cryoprotectants such as propylene glycol, ethylene glycol, glycerol, and ethanol have also been reported. However, these organic solvent cryoprotectants... in vivo It may also exhibit toxicity when administered.

[0006] Therefore, the present invention provides a composition for cell cryopreservation, a cell freezing method, and cell preparations obtained therefrom, which, for example, can protect cells during cell freezing even without substantially adding the aforementioned organic solvent-based cell cryoprotectants.

[0007] Methods for solving problems To achieve the above objectives, the cell cryopreservation composition of the present invention (hereinafter also referred to as the "composition") comprises sugars or their derivatives and / or surfactants.

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

[0009] The cell preparations of the present invention comprise cells and the composition of the present invention.

[0010] The effects of the invention According to the present invention, cells can be protected during cell freezing, for example, even without the substantial addition of organic solvent-based cell cryoprotectants. Attached Figure Description

[0011] Figure 1 This is a graph showing the cooling curve of the cell cryopreservation solution in Example 10.

[0012] Figure 2 This is a graph showing the cell recovery rate of AD-MSCs after thawing under various freezing methods in Example 10.

[0013] Figure 3 This is a graph showing the survival rate of AD-MSCs after freeze-thaw cycles under various freezing methods in Example 10.

[0014] Figure 4 This is a graph showing the number of viable cells of AD-MSCs after culture under various freezing methods in Example 10.

[0015] Figure 5 This is a graph showing the live cell recovery rate of AD-MSCs after thawing under various thawing methods in Example 11.

[0016] Figure 6 This is a histogram representing the particle distribution in Example 14.

[0017] Figure 7 This is a graph representing the relative expression levels of CD9 / CD63 in Example 14. Detailed Implementation

[0018] <Definition> In this invention, "sugar" refers to any sugar. The sugar can be, for example, a monosaccharide, disaccharide, trisaccharide, or a polysaccharide. In this invention, "sugar derivative" refers to a compound in which a sugar has been modified with a modifying group. The sugar derivative is, for example, a glycoside.

[0019] In this invention, "reducing sugar" refers to sugar that has reducing properties, that is, sugar that has a free aldehyde group or a free ketone group. Conversely, "non-reducing sugar" refers to sugar that does not exhibit reducing properties, that is, sugar that does not have a free aldehyde group or a free ketone group.

[0020] In this specification, "glutathione" is a tripeptide composed of glutamic acid, cysteine, and glycine. Glutathione is known to function as an antioxidant within cells, protecting them from damage caused by reactive oxygen species such as free radicals and peroxides.

[0021] In this specification, "amino acid" refers to an organic compound containing both amino and carboxyl groups. The amino acid may also be an organic compound containing both amino and sulfonyl groups. The amino acid may be D-type, L-type, or a mixture of both.

[0022] In this specification, "substantially free" means free of concentrations above the detection limit, or completely free of. Specifically, compositions that do not contain serum or plasma are serum-free and substantially free of serum or plasma. "Serium-free" means free of serum or plasma-derived components at concentrations above the detection limit, or completely free of serum or plasma-derived components. Compositions that do not contain animal-derived components are animal-free and substantially free of human and animal-derived components. "Animal-free" means free of animal-derived components at concentrations above the detection limit, or completely free of animal-derived components. Compositions that do not contain protein are protein-free and contain no protein. "Protein-free" means free of protein at concentrations above the detection limit, or completely free of protein. Compositions that do not contain organic solvent-based cell cryoprotectants are organic solvent-free and substantially free of organic solvent-based cell cryoprotectants. "Organic solvent-free" means free of said organic solvent-based cell cryoprotectants at concentrations above the detection limit, or completely free of said organic solvent-based cell cryoprotectants. Compositions that do not contain albumin are albumin-free and contain no albumin. "Albumin-free" means free of albumin at concentrations above the detection limit, or completely free of albumin.

[0023] In this specification, "organic solvent" refers to an organic compound that is a liquid at room temperature and pressure and has the property of dissolving other substances. The organic compound refers to a compound containing carbon atoms that produces carbon dioxide or undergoes carbonization upon combustion.

[0024] In this specification, “albumin” is a protein known as a component of blood plasma. For example, human serum albumin can be exemplified by proteins having the amino acid sequence registered in GenBank accession number AAN17825.1, or human serum albumin having the corresponding amino acid sequence.

[0025] In this specification, "chemically defined" means that the composition and its content can be defined chemically. Specifically, "chemically defined composition" refers to a composition whose compound name or amino acid sequence is clearly defined and whose content can also be specified.

[0026] In this instruction manual, "infusion" refers to a liquid preparation, injection, or drip that can be administered intravenously.

[0027] In this specification, "culture medium" refers to a culture medium used for cell culture. The culture medium can be prepared, for example, by adding necessary components to a basal culture medium.

[0028] In this specification, "separated" means a state that has been identified and separated, and / or a state that has been recovered from components in their natural state. This "separation" can be achieved, for example, through at least one purification step.

[0029] In this specification, "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. In this specification, "peptide" refers to a polymer composed of unmodified amino acids (natural amino acids), modified amino acids, and / or artificial amino acids. The peptide is a polymer composed of fewer than 10 amino acids.

[0030] In this specification, "cell preparation" refers to a composition containing the desired cells.

[0031] In this specification, "mesenchymal cells" refers to cells derived from the mesoderm or neural crest that constitute connective tissue, and / or cells capable of differentiating into said cells. When said mesenchymal cells have the ability to self-replicate, they may also be called mesenchymal stem cells (MSCs).

[0032] In this specification, "immune cell" means the cell that constitutes the immune system, and / or the cell that has the ability to differentiate into said cell.

[0033] In this specification, "freezing point" refers to the temperature at which a target substance changes from a liquid to a solid. The freezing point can be measured as the temperature at which latent heat is released when the target liquid is cooled.

[0034] In this specification, "treatment" refers to therapeutic and / or preventative treatment. "Treatment" refers to the treatment, cure, prevention, suppression, relief, or improvement of a disease, symptom, or disorder, or the cessation, suppression, reduction, or delay of its progression. "Prevention" refers to reducing the likelihood of a disease or symptom occurring, or delaying its onset. "Treatment" can be, for example, treatment of a subject (patient) suffering from a target disease, or treatment of a model animal of the target disease.

[0035] In this specification, "object" means an animal or a cell, tissue, or organ derived from an animal. The object particularly includes humans. The animal includes both humans and non-human animals. Non-human animals include, for example, mammals such as mice, rats, rabbits, dogs, cats, cattle, horses, pigs, monkeys, dolphins, and sea lions. In this specification, "patient" means an individual receiving preventative or therapeutic treatment. The object includes, for example, not only patients but also healthy individuals.

[0036] The sequence information of the proteins or nucleic acids (such as DNA or RNA) that are encoded in this specification can be obtained from protein databases such as Protein Data Bank, UniProt, or GenBank.

[0037] The present invention will be described below by way of example, but the invention is not limited to the examples below and can be implemented in any way. Furthermore, unless otherwise stated, the descriptions in this invention may be referenced interchangeably. Additionally, the symbol “~” used in this specification indicates the numerical or physical quantity preceding or following it. Furthermore, the expressions “A and / or B” in this specification include “A only”, “B only”, and “both A and B”. Also, in the following description, “mol / L” is sometimes written as “M”.

[0038] <Compositions for Cell Cryopreservation> In one aspect, the present invention provides a cell cryopreservation composition that protects cells during freezing even without substantially the addition of organic solvent-based cell cryoprotectants. The cell cryopreservation composition of the present invention comprises sugars or derivatives thereof, surfactants, and / or polymers containing polyoxyethylene chains.

[0039] Through in-depth research, the inventors have discovered that the sugar or its derivatives, surfactants, and / or polymers containing polyethylene oxide chains can function as cell cryoprotectants. It is speculated that, particularly by achieving a certain concentration or higher of the sugar or its derivatives, surfactants, and / or polymers containing polyethylene oxide chains, the formation of ice crystals inside and outside the cell can be inhibited, and intracellular water can be dehydrated through the osmotic pressure difference between the inside and outside of the cell, thereby protecting the cell membrane and organelles from cryoprotection. However, this speculation does not constitute any limitation to the present invention. Therefore, according to the present invention, by using the sugar or its derivatives, surfactants, and / or polymers containing polyethylene oxide chains as cell cryoprotectants, cells can be protected during cell freezing, for example, even without using organic solvent-based cell cryoprotectants.

[0040] The compositions of the present invention, for example as cell cryoprotectants, comprise the sugar or its derivatives, a surfactant, and / or a polymer containing a polyoxyethylene chain. The compositions of the present invention preferably comprise the sugar or its derivatives because they have a high cell cryoprotective effect. The compositions of the present invention, as cell cryoprotectants, can be used alone or in combination of two or more ingredients. When the compositions of the present invention comprise two or more components, for example, the compositions of the present invention may comprise at least one of the sugar or its derivatives, the surfactant, and the polymer containing the polyoxyethylene chain.

[0041] The sugar is not particularly limited and may include monosaccharides, disaccharides, trisaccharides, polysaccharides, etc. Because cell proliferation rates are higher in culture after thawing, for example, the sugar is preferably a monosaccharide, disaccharide, or trisaccharide, more preferably a monosaccharide or disaccharide. One type of sugar may be used alone, or two or more types may be used in combination.

[0042] The monosaccharides include, for example, glucose, galactose, mannose, fructose, xylose, ribose, arabinose, etc., with glucose being preferred. The disaccharides include, for example, sucrose, maltose, sucralose, lactose, cellobiose, lactobionic acid, lactulose, melibiose, and isomaltose, with sucrose or maltose being preferred, and sucrose being more preferred. The trisaccharides include, for example, raffinose, maltotriose, isomalttriose, panose, pinotriose, etc., with raffinose being preferred. The polysaccharides include, for example, dextran, starch, hydroxyethyl starch, hyaluronic acid, glycogen, alginic acid, etc.

[0043] The sugar can be either a non-reducing sugar or a reducing sugar, but a non-reducing sugar is preferred due to its higher cell cryoprotection effect. Examples of non-reducing sugars include sucrose, trehalose, sucralose, maltotriose, isomalttriose, and panose.

[0044] The sugar derivatives include, for example, nucleosides, nucleotides, and sugar alcohols. A single sugar derivative may be used, or two or more may be used in combination.

[0045] The nucleosides include, for example, ribonucleosides and deoxyribonucleosides. The ribonucleosides include, for example, uridine, adenosine, guanosine, 5-methyluridine, cytidine, etc., with uridine being preferred due to its high cell cryoprotection effect. The deoxyribonucleosides include, for example, deoxyuridine, deoxyadenosine, deoxyguanosine, thymidine, deoxycytidine, etc., with deoxyuridine being preferred.

[0046] The nucleotides include, for example, ribonucleotides and deoxyribonucleotides. The ribonucleotides include, for example, 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, etc. The deoxyribonucleotides include, for example, 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, etc.

[0047] The sugar alcohols include, for example, sorbitol and xylitol.

[0048] The surfactants include, for example, nonionic surfactants such as polyvinylpyrrolidone (PVP), modified polyalkylene glycols, poloxamer, and polyvinyl alcohol; and amphoteric surfactants such as lecithin (egg yolk lecithin, soybean lecithin); etc. The modified polyalkylene glycol is, for example, a polyalkylene glycol modified by a copolymer of polyvinylcaprolactam blocks and polyvinyl acetate blocks. The polyalkylene glycol modified by the copolymer of polyvinylcaprolactam blocks and polyvinyl acetate blocks is, for example, polyethylene glycol modified by a copolymer of polyvinylcaprolactam blocks and polyvinyl acetate blocks. The modified polyalkylene glycol is, for example, a polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer. The modified polyalkylene glycol is preferably Soluplus (registered trademark). The modified polyalkylene glycol can, for example, be a graft copolymer obtained from (i) N-vinylcaprolactam, (ii) vinyl acetate, and (iii) a polyether. The polyether can be polyethylene glycol. The modified polyalkylene glycol may, for example, be a compound obtained by free radical polymerization of the mixture described in (i) to (iii) above. In this case, the composition of the mixture is: (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) is 100 wt%. The modified polyalkylene glycol may also be a compound represented by the following structural formula (Formula I) or a salt thereof. In the chemical formula, the mass percentages (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 sum of the wt% of l, m, and n is, for example, 100 wt%. The wt% of l, m, and n can also be, for example, about 57 wt%, about 30 wt%, and about 13 wt%, respectively.

[0049] In the 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 can be the average degree of polymerization. The weight-average molecular weight (Mw) of the modified polyalkylene glycol is, for example, 70,000–170,000 g / mol, 80,000–160,000 g / mol, 90,000–140,000 g / mol, 100,000–130,000 g / mol, or 110,000–125,000 g / mol. The weight-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 the compound represented by the chemical formula, where n is 13, m is 30, and l is 57. The l, m, and n of Soluplus may also have the stated wt% or degree of polymerization. The modified polyalkylene glycol can be prepared by methods described in, for example, US 2008 / 0293828 A1, US 2010 / 0204425 A1 or US 2018 / 0305636 A1. Furthermore, the components in (i) to (iii) can also be those described in these documents.

[0050] The poloxamer is a block copolymer composed of polyethylene oxide chains and polypropylene oxide chains, wherein polypropylene oxide chains are connected to polyethylene oxide chains on both sides. The poloxamer is, for example, a polymer represented by formula (II). The poloxamer can be appropriately selected according to its molecular weight. Examples of poloxamers include: Pluronic (registered trademark) F-68 (Poloxamer 188), Pluronic (registered trademark) F-127 (Poloxamer 407), Synperonic (registered trademark) F108 (Poloxamer 338), Poloxamer (registered trademark) 124, Poloxamer 237, etc. In the chemical formula, the degrees of polymerization of x, y, and z are, for example, 2–180, 10–90, and 2–180, respectively. In the chemical formula, poloxamer is more suitable as a cell cryoprotectant when the sum of x and z is relatively large compared to the value of y. The degree of polymerization of x is, for example, 2–180, 20–170, 50–160, 60–150, 70–120, 70–110, or 75–105. The degree of polymerization of y is, for example, 10–90, 15–80, 20–70, or 25–60. The degree of polymerization of z is, for example, 2–180, 20–170, 50–160, 60–150, 70–120, 70–110, or 75–105. The degrees of polymerization of x, y, and z can be the average degree of polymerization. The weight-average molecular weight (Mw) of the poloxamer is, for example, 500–25000 g / mol, 1000–22500 g / mol, 2500–20000 g / mol, 5000–17500 g / mol, or 7500–15000 g / mol.

[0051] The polymer containing polyoxyethylene chains refers to polymers whose polymer structure includes polyoxyethylene (–(CH2–CH2–O). n – where n is an integer greater than or equal to 2) polymers. The polymers containing polyoxyethylene chains include, for example, poloxamer, polyethylene glycol, polyvinylpyrrolidone (PVP), modified polyalkylene glycols, etc. The average molecular weight of the polyethylene glycol is, for example, 300–30000, 1000–20000, 2000–6000, or 4000–6000.

[0052] 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 can be the concentration of a single compound or the total concentration of two or more compounds.

[0053] When the composition of the present invention 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 the 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 can be the concentration of a single sugar or the total concentration of two or more sugars.

[0054] When the composition of the present invention 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 can be the concentration of one sugar alcohol or the total concentration of two or more sugar alcohols.

[0055] The concentration of the surfactant is, for example, 0.001–20% (w / v), 0.0015625–10% (w / v), 0.00625–6.4% (w / v), 0.016–3.2% (w / v), 0.08–1.6% (w / v), 0.8–1% (w / v), 1–20% (w / v), 2.5–15% (w / v), 5–15% (w / v), or 7.5–12.5% ​​(w / v). When 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 surfactant can be the concentration of one surfactant or the total concentration of two or more surfactants.

[0056] The concentration of the polymer containing the polyoxyethylene chain is, for example, 0.001–20% (w / v), 0.0015625–10% (w / v), 0.00625–6.4% (w / v), 0.025–3.2% (w / v), 0.08–1.6% (w / v), 0.8–1% (w / v), 1–20% (w / v), 2.5–15% (w / v), 5–15% (w / v), or 7.5–12.5% ​​(w / v). When the polymer containing the polyoxyethylene chain is PVP or modified polyalkylene glycol, the concentration of the PVP or modified polyalkylene glycol can refer to the foregoing examples. When the polymer containing the polyoxyethylene chain is polyethylene glycol, the concentration of the polyethylene glycol is 0.001–20% (w / v), 0.0015625–10% (w / v), 0.00625–6.4% (w / v), 0.016–3.2% (w / v), 0.08–1.6% (w / v), 0.8–1% (w / v), 1–20% (w / v), 2.5–15% (w / v), 5–15% (w / v), or 7.5–12.5% ​​(w / v). The concentration of the polymer containing the polyoxyethylene chain can be the concentration of one polymer containing the polyoxyethylene chain, or the total concentration of two or more polymers containing the polyoxyethylene chain.

[0057] The compositions of the present invention may, for example, contain antioxidants (reducing agents). These antioxidants are substances capable of inhibiting oxidation reactions. By including these antioxidants, the compositions of the present invention can further improve the cell cryoprotection effect, particularly the cell cryoprotection effect of sugars or sugar derivatives. The antioxidants include, for example, glutathione, lipoic acid (α-lipoic acid, alpha-lipoic acid), ascorbic acid (vitamin C) or its derivatives, tocopherol (vitamin E) or its derivatives, acetylcysteine, and chelating agents. The ascorbic acid derivatives include, for example, ascorbic acid-2-glucoside, ascorbic acid-2-phosphate, tetrahexyldecyl ascorbate, and 3-O-ethyl ascorbic acid. The tocopherol derivatives include, for example, α-tocopherol phosphate, tocopherol acetate, and tocopherol succinate. The chelating agents include, for example, ethylenediaminetetraacetic acid (EDTA), citric acid, gluconic acid, sodium gluconate, and phytic acid. The antioxidants may be used alone or in combination of two or more.

[0058] When the composition of the present invention contains the antioxidant, it can particularly improve the cell cryoprotection effect. More preferably, the antioxidant includes the glutathione, the lipoic acid, the ascorbic acid or a derivative thereof and / or the tocopherol or a derivative thereof, and even more preferably, it includes the lipoic acid and the glutathione.

[0059] Because it can particularly improve the cell cryoprotection effect, the composition of the present invention 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.

[0060] When the composition of the present invention 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, 10 to 100 μmol / L, 1 to 2000 μmol / L, 8 to 500 μmol / L, or 32.5 to 100 μmol / L. The concentration of the antioxidant can be, for example, the concentration of one antioxidant or the total concentration of two or more antioxidants.

[0061] The concentration of the glutathione is, for example, 10 nmol / L to 10 mmol / L, 100 nmol / L to 1 mmol / L, 1 μmol / L to 100 μmol / L, 1 to 2000 μmol / L, 8 to 500 μmol / L, or 32.5 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. The concentration of the ascorbic acid or its derivatives is, for example, 1 nmol / L to 100 mmol / L, 65 nmol / L to 2 mmol / L, 325 nmol / L to 1500 μmol / L, 1 to 1000 μmol / L, 1.625 to 203.125 μmol / L, 1.625 to 1015.625 μmol / L, or 8.125 to 40.625 μmol / L. The concentration of the tocopherol or its derivatives is, for example, 1 nmol / L to 100 mmol / L, 65 nmol / L to 2 mmol / L, 325 nmol / L to 1500 μmol / L, 1 to 1000 μmol / L, 1.625 to 203.125 μmol / L, or 8.125 to 40.625 μmol / L. The concentration of the lipoic acid is, for example, 1 nmol / L to 100 mmol / L, 65 nmol / L to 2 mmol / L, 325 nmol / L to 1500 μmol / L, 1 to 1000 μmol / L, 1.625 to 203.125 μmol / L, 8.125 to 40.625 μmol / L, 77.55 nmol / L to 48.46 μmol / L, 77.55 nmol / L to 9.69 μmol / L, or 387.72 nmol / L to 1.94 μmol / L.

[0062] The compositions of the present invention may also contain amino acids. By containing said amino acids, the compositions of the present invention can further improve the cell cryoprotection effect. The amino acids include, for example, α-amino acids, β-amino acids, γ-amino acids, etc. The amino acids are, for example, amino acids that constitute proteins. The amino acids may also be sulfur-containing amino acids. The amino acids may be used alone or in combination of two or more. The amino acids include, for example, alanine, taurine, phenylalanine, methionine, proline, cysteine, serine, lysine, arginine, homoserine, GABA, aminocaproic acid, tryptophan, and glycine, etc.

[0063] The concentration of the amino acid is, for example, 0.01–100 mmol / L, 0.1–50 mmol / L, or 0.5–10 mmol / L. The concentration of the amino acid can be, for example, the concentration of a single amino acid or the total concentration of two or more amino acids.

[0064] In the compositions of the present invention, each compound may be, for example, a salt. There are no particular limitations on the salts of the compounds, and they may include, for example, inorganic or organic salts. The salts include, for example, metal salts, ammonium salts, salts formed with organic bases, salts formed with inorganic acids, salts formed with organic acids, and salts formed with basic or acidic amino acids. 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 formed with organic bases include, for example, salts formed with trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N'-dibenzylethylenediamine, etc. Salts formed with inorganic acids include, for example, salts formed with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc. The salts formed with organic acids include, for example, salts formed with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, methanesulfonic acid, p-toluenesulfonic acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. The salts formed with basic amino acids include, for example, salts formed with arginine, lysine, ornithine, etc. The salts formed with acidic amino acids include, for example, salts formed with aspartic acid, glutamic acid, etc. The salts include pharmaceutically acceptable salts. Pharmaceutically acceptable salts include, for example, forms having a reasonable benefit suitable for pharmaceutical use.

[0065] The compositions of the present invention can be, for example, liquids or solids. When the compositions of the present invention are liquids, they may further contain, for example, an aqueous solvent.

[0066] The aqueous solvent is, for example, an aqueous solvent used for cells, specifically including water, buffer solutions, infusions, culture media, etc., preferably buffer solutions and infusions. The buffer solution includes, for example, Hanks' balanced salt solution (HBSS), phosphate buffer, etc. The infusion includes, for example, isotonic electrolyte infusions such as bicarbonate Ringer's solution, physiological saline, Ringer's solution, lactated Ringer's solution, acetate Ringer's solution; hypotonic electrolyte infusions such as solution 1 (starting solution), solution 2 (dehydration replenishment solution), solution 3 (maintenance solution), solution 4 (postoperative recovery solution); and amino acid infusions, etc. The culture medium can be, for example, a basal culture medium. The basal culture medium includes, for example, MEM medium (Gibco Invitrogen, etc.), MEMα medium (Gibco Invitrogen, etc.), DMEM medium (Gibco Invitrogen, etc.), IMDM medium (Wako Pure Chemical, etc.), RPMI 1640 medium (Gibco Invitrogen, etc.), Ham F-12 medium (Gibco, etc.), RD medium, or a mixture of these media.

[0067] The compositions of the present invention, for example, can achieve 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 freeze-thaw experiment. Furthermore, the compositions of the present invention, for example, can achieve cell viability of 60-100%, 65-99%, 70-99%, 75-98%, 80-97%, 85-96%, or 90-95% after a freeze-thaw experiment. The cell freeze-thaw experiment can be performed using the same method as in Example 1 described later. Specifically, 1×10 ５ ~2×10 6 Add 0.5 mL of target cell cryopreservation solution to each adipose-derived stromal cell (AD-MSC) and suspend it. Then, rapidly freeze the resulting cell suspension at -80°C. For the frozen AD-MSCs, thaw the tubes in a water bath at approximately 25°C starting the day after freezing. After thawing, wash the AD-MSCs with HBSS(+), recover the cells from the tubes, centrifuge, remove the supernatant, and suspend the AD-MSCs in culture medium. Add trypan blue to the resulting cell suspension, mix, and drop a portion of the mixture onto a glass slide. Count the number of viable cells using a cell counter. Subsequently, the cell viability can be determined by the number of viable cells after freezing (N). b ) as a percentage of the total number of cells after freezing (N) a The proportion of (N) b / N a The result is calculated as (×100 (%)).

[0068] The compositions of the present invention are preferably substantially free of organic solvent-based cell cryoprotectants. Such organic solvent-based cell cryoprotectants include, for example, dimethyl sulfoxide, propylene glycol, ethylene glycol, glycerol, and ethanol. The compositions of the present invention are preferably substantially free of dimethyl sulfoxide, propylene glycol, ethylene glycol, glycerol, and ethanol.

[0069] The compositions of the present invention preferably contain substantially no animal-derived ingredients. These animal-derived ingredients include, for example, proteins, lipids, peptides, hormones, amino acids, vitamins, etc. The compositions of the present invention preferably contain substantially no animal-derived proteins, lipids, peptides, hormones, amino acids, and / or vitamins; more preferably, they contain substantially no animal-derived proteins, lipids, peptides, hormones, amino acids, and vitamins.

[0070] The compositions of the present invention are preferably substantially free of serum and / or plasma. More preferably, the compositions of the present invention are free of said serum and plasma.

[0071] The compositions of the present invention are preferably chemically defined compositions, for example.

[0072] The compositions of the present invention do not contain, for example, polymers composed of amino-containing units, or polymers partially or wholly blocked by carboxylic anhydrides. The amino-containing polymers are selected, for example, from ε-poly-L-lysine, α-poly-L-lysine, polyarginine, other polyamino acids, amino-modified polysaccharides, and polyallylamine. The carboxylic anhydrides include, for example, acetic anhydride, citric anhydride, succinic anhydride, glutaric anhydride, malic anhydride, fumaric anhydride, and maleic anhydride.

[0073] The compositions of the present invention do not contain, for example, polymers composed of carboxyl-containing units, or polymers in which said polymers are partially or wholly blocked by compounds having multiple amino groups. The polymers are selected, for example, from polyacrylic acid, α-polyglutamic acid, γ-polyglutamic acid, polyglutamic acid, polyaspartic acid, other polyamino acids, and carboxylated polysaccharides. The compounds having multiple amino groups may include diamines, such as ethylenediamine, adipic acid dihydrazides, etc.; triamines; polyamines, etc.

[0074] In the compositions of this invention, the target cell can be any cell. The cells include, for example, cells cultured in vitro, primary cultured cells, cells isolated from an organism, cultured cell lines, etc. As specific examples, the cells include, for example, mesenchymal cells, immune cells, etc. Mesenchymal cells are typically found inside and around organs such as blood vessels, liver, or pancreas, in adipose tissue, bone marrow, or umbilical cord. The mesenchymal stem cells are a type of pluripotent stem cell, referring to cells capable of differentiating into adipocytes, osteocytes, chondrocytes, myocytes, hepatocytes, tendon cells, and / or nerve cells. Depending on the tissue from which they are collected, the mesenchymal stem cells may also be referred to as adipose tissue-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placental-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, and umbilical cord-derived mesenchymal stem cells, etc. The immune cells include, for example, T cells, B cells, NK cells, NKT cells, monocytes, macrophages, dendritic cells, etc. In addition to these cells, the cells may also be skin cells, keratinocytes, skeletal muscle cells, cardiomyocytes, lung cells, mesenteric cells, adipocytes, stem cells, hepatocytes, epithelial cells (epithelial-like cells), Kupffer cells, fibroblasts, neurons, cardiomyocytes, myocytes, 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-secreting cells, etc.

[0075] The cells may also be tissues or organs composed of multiple cells. These tissues include, for example, epithelial tissues such as skin and intestinal epithelium; muscle tissues such as connective tissue, skeletal muscle, and cardiac muscle; and neural tissues such as the brain, spinal cord, and nerves.

[0076] The cells may also be cell-derived components. These cell-derived components include, for example, cell culture supernatant, exosomes (EVs, extracellular vesicles), microvesicles, apoptotic bodies, etc. When the cell-derived component is cell culture supernatant and / or exosomes, the source of the exosomes is, for example, the cells. As a specific example, the cell culture supernatant and / or exosomes may be cell culture supernatant and / or exosomes derived from adipose tissue, bone marrow, or umbilical cord mesenchymal stem cells.

[0077] The compositions of the present invention, for example, when the composition is a solid, can be prepared by mixing the components. The compositions of the present invention, for example, when the composition is a liquid, can be prepared by mixing the components and the aqueous solvent.

[0078] The compositions of the present invention are suitable for cryopreservation of the cells, for example, even without the use of the organic solvent-based cryoprotectant.

[0079] <Cell freezing methods> On the other hand, the present invention provides a cell freezing method that can protect cells during freezing even without substantially adding the aforementioned organic solvent-based cell cryoprotectant. The cell freezing method of the present invention includes a freezing step of freezing cells in the presence of the cell cryopreservation composition of the present invention.

[0080] In the following description, unless otherwise stated, the description of the concentration of each component in the composition of the present invention also applies to the description of the concentration of each component in the freezing process.

[0081] The freezing method of the present invention may further include, for example, a step of contacting the composition of the present invention with the cells prior to the freezing step. This contact may be achieved, for example, by mixing the cells with the composition of the present invention or by suspending the cells in the composition of the present invention. The resulting cell-containing cryopreservation solution has a cell concentration, for example, of 1 × 10⁻⁶. 2 ~1×10 11 cells / mL, 1×10 3 ~1×10 10 Cells / mL or 1×10 4 ~1×10 9 Cells / mL.

[0082] Next, in the freezing process, for example, the cryopreservation solution (cell preparation) containing the cells is frozen. The freezing can be performed using conventional freezing methods for cell freezing. The freezing can be rapid freezing or slow freezing, but rapid freezing is preferred because it improves the cell viability or recovery rate after thawing. Furthermore, during the freezing process, the cryopreservation solution can be frozen by continuous cooling or non-continuous cooling, but continuous cooling is preferred because it improves the cell viability or recovery rate after thawing. As specific examples, the freezing can be performed by placing the cryopreservation solution containing the cells into test tubes and freezing them in a freezer at -20°C, -40°C, or -80°C; the test tubes can also be frozen in the gas or liquid phase of liquid nitrogen; or they can be frozen in a freezing device such as a proton freezer. In the freezing process, 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. The cooling rate can be adjusted, for example, by changing the type of freezing method or by using a programmed cooling system. 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.

[0083] It is generally believed that the survival rate of cells after freeze-thaw can be regulated by controlling the formation and growth of ice crystals of water inside and outside the cells during the freezing process. Furthermore, these ice crystals typically form near the freezing point, primarily between the freezing point and -40°C, and especially between the freezing point and -20°C. The inventors have discovered that when cells are frozen using the composition of this invention, compared to using cell cryoprotectants containing the aforementioned organic solvents, faster cooling during cell freezing, particularly within the ice crystal formation temperature range, i.e., a more rapid decrease in temperature, can further improve the survival rate or recovery rate of cells after thawing. Therefore, in the freezing process, the rate of temperature change of the composition as it passes its freezing point is, for example, -0.5 to -250°C / min, -0.624 to -200°C / min, -0.5 to -150°C / min, -0.5 to -100°C / min, -0.5 to -90°C / min, -0.5 to -80°C / min, -0.5 to -70°C / min, -0.624 to -58.8°C / min, -1 The temperature change rates are: -42°C / min, -1 to -40°C / min, -1 to -35°C / min, -1 to -30°C / min, -1.105 to -25.782°C / min, -3.429 to -25°C / min, -3.691 to -20°C / min, -3.925 to -15°C / min, -4.18 to -15°C / min, -4.384 to -10°C / min, or -5.294 to -10°C / min. To further improve cell viability or recovery rate after thawing, the temperature change rate of the composition through the freezing point is more preferably -3 to -60°C / min, -4 to -55°C / min, -5 to -50°C / min, -6 to -45°C / min, or -7 to -40°C / min. The rate of temperature change when passing the freezing point is the average temperature change of the cell preparation before supercooling occurs, within the range of -1℃ to +1℃, with the freezing point as the reference. The rate of temperature change when passing the freezing point can be calculated using the same method as in Example 10 described later.

[0084] In the freezing process, the average cooling rate of the composition in the range of 0 to -20°C is, for example, -0.4 to -100°C / min, -0.447 to -75°C / min, -0.5 to -50°C / min, -0.736 to -25°C / min, -1 to -25°C / min, -1.333 to -22.526°C / min, -1.5 to -20°C / min, -1.5 to -15°C / min, -1.951 to -14.966°C / min, -2.073 to -14°C / min, -2.094 to -13°C / min, -2.284 to -12°C / min, -2.673 to -11°C / min, -3 to -10°C / min, -3.458 to -9°C / min, or -4 to -8.685°C / min. The average cooling rate can be calculated using the same method as in Example 10 described later.

[0085] In the freezing process, the cell preparation may also be freeze-dried. The freeze-drying process can be performed using known freeze-drying methods, and specifically includes a freezing process to freeze the cell preparation and a drying process to dry the frozen cell preparation. First, in the freezing process, the temperature or ambient temperature of the cell preparation is adjusted to below -30°C, -40°C, -50°C, -60°C, -70°C, or -80°C. This temperature adjustment can be achieved, for example, by contact with liquid nitrogen or by storage in a freezer. The freezing time is not particularly limited and can be set according to the type of temperature adjustment process; it can be instantaneous or a pre-set time, specifically, for example, 0–24 hours or 6–24 hours. The freezing process is performed, for example, at the stated temperature for 6–24 hours to ensure that the cell preparation is reliably frozen. The drying process can be performed, for example, using vacuum freeze-drying. The temperature conditions for vacuum freeze-drying are not particularly limited and can be set according to the cell type and liquid volume in the cell preparation. As a specific example, the upper limit of the temperature conditions is not particularly limited, and can be, for example, below -30°C, below -35°C, below -40°C, or below -45°C. The lower limit of the temperature conditions is not particularly limited, and can be, for example, above -80°C, above -75°C, above -70°C, above -65°C, above -60°C, above -55°C, or above -50°C. The temperature conditions are, for example, -80 to -30°C, -75 to -30°C, -70 to -35°C, -65 to -35°C, -60 to -40°C, -55 to -40°C, or -50 to -45°C. The pressure conditions for the vacuum freeze-drying process are not particularly limited, and can be set according to the cell type and liquid volume in the cell preparation. The upper limit of the pressure conditions is not particularly limited, and can be, for example, below 20 Pa, below 18 Pa, below 16 Pa, or below 15 Pa. There is no particular limitation on the lower limit of the gas pressure condition, for example, it can be above 0 Pa, greater than 0 Pa, above 1 Pa, above 2 Pa, above 3 Pa, above 4 Pa, or above 5 Pa. The gas pressure condition can be, for example, 0–20 Pa, greater than 0 Pa but not exceeding 20 Pa, 1–20 Pa, 2–18 Pa, 3–18 Pa, 4–16 Pa, or 5–15 Pa. There is no particular limitation on the time of the vacuum freeze-drying process, for example, it can be set according to the volume of the cell preparation. There is no particular limitation on the lower limit of the time condition, for example, it can be above 1 hour, above 2 hours, above 3 hours, above 4 hours, above 5 hours, above 6 hours, above 7 hours, or above 8 hours.There is no particular upper limit to the time condition, such as less than 72 hours, less than 48 hours, less than 24 hours, less than 20 hours, less than 15 hours, less than 10 hours, or less than 8 hours. The time condition can be, for example, 1 to 72 hours, 2 to 48 hours, 3 to 24 hours, 4 to 20 hours, 5 to 15 hours, 6 to 10 hours, or 7 to 8 hours.

[0086] The freezing method of the present invention may further include, for example, a preservation step of storing the frozen cells in a frozen state. The preservation may be carried out, for example, by placing a cryopreservation solution containing the cells in a freezer or in a liquid nitrogen atmosphere. The preservation temperature in the preservation step may be, for example, -20 to -196°C, -40 to -150°C, or -70 to -90°C. The preservation period in the preservation step may be any period, specifically, for example, 1 day to 5 years, 10 days to 3 years, or 1 to 2 years.

[0087] The freezing method of the present invention may further include, for example, a thawing step (thawing step) for thawing the cryopreserved cells. The thawing step may be carried out, for example, by heating the cryopreservation solution containing the cells. The heating may be carried out at, for example, 20–80°C, 20–60°C, 20–40°C, 30–60°C, 30–37°C, 35–45°C, 38–42°C, or about 40°C, i.e., the cell preparation is placed under any temperature condition for heating. Furthermore, it is generally believed that ice crystals formed mainly between -10°C and -40°C during the thawing process of the frozen cells can damage the cells. The inventors have discovered that when the cell preparation is thawed using the composition of the present invention, compared to the use of a cell cryoprotectant containing the organic solvent, faster heating, i.e., a more rapid increase in temperature, can further improve the survival rate or recovery rate of the thawed cells. Therefore, the heating is preferably carried out at temperatures of 38–80°C, 38–70°C, 38–60°C, 38–50°C, 39–45°C, or 39–41°C. The heating can be carried out, for example, by placing the container containing the cell preparation in a hot water bath within the specified temperature range, or by using a heating block.

[0088] In the thawing process, thawing is preferably performed in the presence of a cell resuscitation solution (thawing fluid), as this improves the survival rate or recovery rate of the thawed cells. When using the resuscitation solution, it can be added to the cell preparation containing the cells, for example, at the beginning of thawing or during the thawing process. The resuscitation solution can be, for example, an aqueous solvent such as water, buffer solution, infusion fluid, or culture medium. The temperature of the resuscitation solution is, for example, 0–45°C, 35–40°C, or approximately 37°C.

[0089] The volume ratio (L:C) of the resuscitation solution (thawing solution) (L) to the cell preparation (C) is, for example, 1:0.1–100, 1:0.3–50, or 1:0.5–20. The volume ratio (C:L) of the cell preparation (C) to the resuscitation solution (thawing solution) (L) is, for example, 1:0.1–100, 1:0.3–50, or 1:0.5–20.

[0090] The freezing method of the present invention can, for example, achieve a cell viability rate 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 the thawing process. The freezing method of the present invention can, for example, achieve a cell viability rate of 60-100%, 65-99%, 70-99%, 75-98%, 80-97%, 85-96%, or 90-95% after the thawing process. The cell viability rate after the thawing process can, for example, be calculated using the same method as for the cell viability rate after the aforementioned freeze-thaw experiment.

[0091] The freezing method of the present invention can, for example, achieve a cell recovery rate 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 the thawing step. The freezing method of the present invention can, for example, achieve a cell recovery rate of 60-100%, 65-99%, 70-99%, 75-98%, 80-97%, 85-96%, or 90-95% after the thawing step. The recovery rate after the thawing step can, for example, be calculated based on the number of viable cells (N) obtained after the thawing step. a ) relative to the number of cells used for freezing (N) b The proportion of (N) a / N b Calculate using ×100(%).

[0092] The freezing method of the present invention can be performed, for example, in vitro or in vivo.

[0093] The freezing method of the present invention is suitable for cryopreservation of cells, for example, even without the use of the organic solvent-based cryoprotectant.

[0094] <Cell Preparations> In another aspect, the present invention provides a cell preparation that substantially does not contain the aforementioned organic solvent-based cell cryoprotectant. The cell preparation of the present invention comprises cells and the cell cryopreservation composition of the present invention.

[0095] The description of the concentrations of each component in the compositions of the present invention is equally applicable, for example, to the description of the concentrations of each component in the cell preparations of the present invention.

[0096] The cell preparations of the present invention can be in a frozen state, i.e., a frozen state or a solid state; or in a pre-freezing or freeze-thawed state, i.e., a liquid state. In the latter case, the cell preparations of the present invention are, for example, in a state where the cells are present in the composition of the present invention. The cell preparations of the present invention are preferably in a frozen state or a solid state. The solid state can be, for example, a lyophilized state. In this case, the cell preparations can also be referred to as a lyophilized composition of cell preparations.

[0097] The cell preparations of the present invention can be obtained, for example, by the cell freezing method of the present invention.

[0098] Since the cell preparations of the present invention are substantially free of the aforementioned organic solvent-based cell cryoprotectants, they can suppress the toxicity caused by organic solvents. Therefore, the cell preparations of the present invention are suitable for use, for example, as cell-based drugs.

[0099] <Thawing methods for cell preparations> In another aspect, the present invention provides a method for thawing cell preparations that are substantially free of the aforementioned organic solvent-based cell cryoprotectants. The thawing method for cell preparations of the present invention (hereinafter also referred to as the "thawing method") includes a thawing step of thawing a frozen cell preparation, wherein the cell preparation is the cell preparation of the present invention. According to the thawing method of the present invention, for cell preparations that are substantially free of the aforementioned organic solvent-based cell cryoprotectants, the survival rate or recovery rate of cells after thawing can be improved.

[0100] In the thawing method of the present invention, the frozen cells are, for example, cell preparations comprising cells obtained in the freezing or preservation step of the freezing method of the present invention and a cryopreservation composition.

[0101] In the thawing method of the present invention, the thawing process can be described, for example, with reference to the thawing process in the freezing method of the present invention.

[0102] <Disposal Methods> In another embodiment, the present invention provides a disposal method that reduces the toxicity caused by the organic solvent-based cell cryoprotectant. The disposal method of the present invention includes an administration step of applying the cell preparation of the present invention to a subject.

[0103] In this invention, the object is, for example, a disease patient who can be treated with the cells contained in the cell preparation. The treatment method of this invention can be carried out, for example, in vitro or in vivo.

[0104] The cell preparations of the present invention, for example, can protect cells during cell freezing without substantially containing the aforementioned organic solvent-based cryoprotectants. Therefore, according to the method of treatment of the present invention, since the amount of the organic solvent-based cryoprotectant applied to the object can be reduced, the toxicity caused by the organic solvent-based cryoprotectant can be reduced.

[0105] Example The embodiments of the present invention will now be described. However, the present invention is not limited to the following embodiments. Unless otherwise stated, commercially available reagents should be used in accordance with their operating procedures. Furthermore, in the following description of the embodiments, "mol / L" is sometimes also referred to as "M".

[0106] [Reagents] In the following examples, unless otherwise stated, each reagent uses the following reagents.

[0107] Table 1 Table 2 [Solvent] In the following embodiments, the solvent used for the preservation solution has the composition described in Tables 3 and 4 below.

[0108] Table 3 Table 4 [Cell Preparation] The cells used in the following examples were prepared as follows.

[0109] (1) Self-made adipose-derived stromal cells (adipose-derived mesenchymal stem cells (AD-MSCs)) Adipose-derived stromal cells were prepared from fresh adipose tissue (obtained via StemExpress). The adipose tissue was washed with HBSS(+), and an equal volume of collagenase solution (Solallis Bio Inc.) was added to the adipose tissue, followed by collagenase treatment at 37°C for 3.5 hours. The resulting solution was then centrifuged (800 g × 5 min). The supernatant containing oil was discarded. The precipitate was washed with the obtained HBSS(+), and the centrifugation and washing were repeated twice. Red blood cells were then lysed using erythrocyte lysis buffer (Solallis Bio Inc.). The lysis buffer was then centrifuged (800 g × 5 min). The supernatant was then discarded. The resulting precipitate (tissue fragments) was resuspended in culture medium (Solallis Bio Inc., M101-AF-500). The tissue fragments were then filtered through a cell sieve. The resulting filtrate was used as a cell suspension containing SVF (Stromal Vascular Fraction), and the solvent was replaced with cell cryopreservation medium (Solallis Bio Inc., C101-AF-100). The cells were then cryopreserved at -80°C. The cryopreserved SVF was washed with HBSS(+). The SVF was then resuspended in culture medium. The resuspended liquid was then seeded into T-25 culture flasks (Sumitomo Bakelite Corporation, MS-23050) and cultured. The seeded cells were collected and cryopreserved at -80°C. The cryopreserved cells were then expanded in T-150 culture flasks (Sumitomo Bakelite Corporation, MS-23600) using culture medium. After expansion culture, when the cells reached a near-confluence state, the AD-MSCs were recovered.

[0110] (2) Prepared from adipose-derived stromal cells (commercially available AD-MSCs) Adipose-derived stromal cells (manufactured by Lonza, PT-5006) were expanded and cultured using T-150 culture flasks (manufactured by Sumitomo Bakelite Corporation, MS-23600). After the expansion culture, when the cells reached a near-confluence state, the AD-MSCs were recovered.

[0111] (3) Preparation of umbilical cord-derived mesenchymal cells (UC-MSCs) Except for replacing the commercially available AD-MSCs with umbilical cord-derived stromal cells (manufactured by LIFELINE, FC-0020), the process was the same as in (2), and the UC-MSCs were recovered.

[0112] (4) Preparation of THP-1 cells Except for replacing the commercially available AD-MSCs with THP-1 cells (manufactured by ATCC, TIB-202), replacing the culture medium for the AD-MSCs with RPMI-1640 + 10% FBS (manufactured by Gibco, 72400047 and A31604-01), and replacing the T-150 culture flasks with T-25 non-adherent culture flasks (manufactured by Sumitomo Bakelite Corporation, MS-2305R), the rest was the same as in (2), and the THP-1 cells were recovered.

[0113] (5) Preparation of Jurkat cells Except for replacing the THP-1 cells with Jurkat cells (manufactured by KAC Corporation, EC88042803-G0), the process is the same as in (4), and the Jurkat cells are recovered.

[0114] [Example 1] It was confirmed that using a cell cryopreservation solution containing sucrose can improve cell viability during cryopreservation.

[0115] (Cryopreservation experiment) The self-made AD-MSCs were seeded into T-150 culture flasks containing culture medium and cultured for 3–4 days. Once the AD-MSCs had proliferated to a near-confluence state, a dissociation agent (Solallis Bio Inc., D101-AF-500) was added to the T-150 culture flasks, and the mixture was incubated at 37°C for 5 minutes. The AD-MSCs were then dissociated and centrifuged (400×g, 5 minutes), and the supernatant was removed. The recovered AD-MSCs were resuspended 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 (Thermo Fisher Scientific, Countess 3). Next, the AD-MSCs were divided into tubes containing 1×10⁶ cells each. 5 ~2×10 6 Cells were aliquoted into test tubes. After aliquoting, the AD-MSCs were centrifuged (400×g, 5 min) and the supernatant was removed. Then, 0.5 mL of the cell cryopreservation solutions shown in Table 5 were added to each test tube and the cells were resuspended. The resulting cell suspensions were then rapidly frozen in an ultra-low temperature freezer (-80°C). DMSO was typically used as a cell cryoprotectant as a positive control. Additionally, the group using only HBSS(+) did not contain a cell cryoprotectant and served as a negative control.

[0116] Table 5 For frozen AD-MSCs, the tubes were thawed in a water bath at room temperature (approximately 25°C) the day after freezing began. After thawing, the AD-MSCs were washed with HBSS(+), recovered from the tubes, and centrifuged (400×g, 5 min). After centrifugation, the supernatant was removed, and the AD-MSCs were resuspended in the culture medium. Trypan blue was added to the resulting cell suspension, and a portion of the mixture was dropped onto a glass slide. The number of viable cells was counted using the cell counter. Then, the total number of cells after cryopreservation (N) was determined. b ) and the number of live cells after cryopreservation (N) a ), calculate cell viability (N) a / N b ×100(%). These results are shown in Table 6 below.

[0117] Table 6 Table 6 shows the results of cell viability after freeze-thaw of AD-MSCs (adipose-derived mesenchymal stem cells). As shown in Table 6, the cell viability after freeze-thaw was significantly reduced in the negative control group (HBSS+ only). In contrast, the cell viability was higher in the 10% sucrose and 10% DMSO groups than in the HBSS+-only group. That is, it was found that the use of sucrose can improve cell viability during freezing, meaning that sucrose can function as a cell cryoprotectant.

[0118] [Example 2] It was confirmed that using cell cryopreservation solutions containing different concentrations of sucrose can improve cell viability during cryopreservation.

[0119] Except for replacing the cell cryopreservation solution shown in Table 5 with the cell cryopreservation solution shown in Table 7 below, the rest was the same as in Example 1, and the survival rate of AD-MSCs after freeze-thaw was calculated. These results are shown in Table 8 below.

[0120] Table 7 Table 8 Table 8 shows the results of AD-MSC cell viability after freeze-thaw cycles. As shown in Table 8, cell viability improved after freeze-thaw at any sucrose concentration. In particular, by increasing the sucrose concentration to 5% or higher, cell viability reached approximately 70%, demonstrating a high viability. Generally, a cell cryopreservation solution with a viability exceeding 70% after freeze-thaw is considered to have excellent performance. Therefore, it can be seen that increasing the sucrose concentration to 5% or higher significantly improves cell viability after freeze-thaw.

[0121] [Example 3] It was confirmed that using cell cryopreservation solutions containing antioxidants (reducing agents) can improve cell viability during cryopreservation.

[0122] Except for replacing the cell cryopreservation solutions shown in Table 5 with those shown in Tables 9-11 below, the process was the same as in Example 1, and the survival rate of AD-MSCs after freeze-thaw was calculated. In Table 13 below, the survival rate (N) without glutathione (sample 8) is used as the benchmark. c Based on the baseline, the survival rate (N) for each glutathione concentration (samples 9–14) was calculated. d ) Calculate the relative survival rate (N) d / N c ×100(%). Furthermore, in Table 14 below, the number of viable cells (N) without the addition of α-lipoic acid (sample 15) is used. E The number of viable cells (N) at different concentrations of α-lipoic acid (sample 16) and α-lipoic acid addition. f ), calculate the relative viable cell count (N) f / N E ×100(%). These results are shown in Tables 12–14 below.

[0123] Table 9 Table 10 Table 11 Table 12 Table 13 Table 14 Table 12 shows the results of AD-MSC viability. Table 13 shows the results of AD-MSC relative viability. Table 14 shows the results of AD-MSC relative viable cell count. As shown in Table 12, the cell viability after freeze-thaw was improved by using any antioxidant. In particular, by setting the glutathione concentration to 32.5 μM, the cell viability reached approximately 86%, demonstrating a high viability. That is, using glutathione as an antioxidant can further improve the cell viability after thawing. Furthermore, as shown in Table 13, compared with no glutathione addition, the relative cell viability after freeze-thaw was improved by adding glutathione. That is, it can be seen that setting the glutathione addition concentration to 1 μM or higher can improve the cell viability during cryopreservation. Further, as shown in Table 14, compared with adding only glutathione as an antioxidant, the relative viable cell count after freeze-thaw was increased by adding both glutathione and α-lipoic acid. That is, it can be seen that by using glutathione and alpha-lipoic acid as antioxidants, the survival rate of cells after freeze-thaw can be improved, and glutathione and alpha-lipoic acid can play the role of antioxidants in cell cryoprotection.

[0124] [Example 4] It was confirmed that using cell cryopreservation solutions containing sugars, sugar alcohols, nucleotides, PVP, or Soluplus can improve cell survival during cryopreservation.

[0125] Except for replacing the cell cryopreservation solutions in Table 5 with those in Tables 15-17 below, the process was the same as in Example 1, and the survival rate of AD-MSCs after freeze-thaw was calculated. These results are shown in Tables 18-20 below.

[0126] Table 15 Table 16 Table 17 Table 18 Table 19 Table 20 Tables 18-20 show the results of AD-MSC survival rates after freeze-thaw cycles. As shown in Tables 18-20, not only sucrose, but also the addition of other disaccharides, monosaccharides, trisaccharides, polysaccharides, sugar alcohols, nucleosides, PVP, and Soluplus can improve cell survival rates after freeze-thaw cycles. That is, it can be seen that disaccharides, monosaccharides, trisaccharides, polysaccharides, sugar alcohols, nucleosides, PVP, and Soluplus can all function as cell cryoprotectants.

[0127] [Example 5] It was confirmed that using solvents other than HBSS in the cell cryopreservation solution can improve cell viability during cryopreservation.

[0128] Except for replacing the cell cryopreservation solution in Table 5 with the cell cryopreservation solution in Table 21 below, the rest was the same as in Example 1, and the survival rate of AD-MSCs after freeze-thaw was calculated. These results are shown in Tables 22 and 23 below.

[0129] Table 21 Table 22 Table 23 Tables 22 and 23 show the results of AD-MSC survival rates after freeze-thaw cycles. As shown in Tables 22 and 23, cell survival rates improved not only in HBSS+ but also in other solvents. That is, for cell cryopreservation solutions containing sucrose and glutathione, cell survival rates during cryopreservation can be improved not only in HBSS+ but also in other solvents.

[0130] [Example 6] It was confirmed that various freezing methods can improve cell viability during cryopreservation in cell cryopreservation solutions containing sucrose and glutathione.

[0131] Except for replacing the cell cryopreservation solution in Table 5 with the cell cryopreservation solution in Table 24 below, and replacing the freezing method with rapid freezing in an ultra-low temperature freezer (-80°C) with the freezing method in Table 25 below, the rest was the same as in Example 1, and the survival rate of AD-MSCs after freeze-thaw was calculated. These results are shown in Table 25 below.

[0132] Table 24 Table 25 Table 25 shows the results of AD-MSC survival rates after freeze-thaw. As shown in Table 25, cell survival rates were improved regardless of the freezing method used. That is, it can be seen that various freezing methods can improve cell survival rates during cryopreservation in cell cryopreservation solutions containing sucrose and glutathione.

[0133] [Example 7] It was confirmed that cell viability during cryopreservation can be improved in cell cryopreservation solutions containing sucrose and amino acids.

[0134] Except for replacing the cell cryopreservation solution in Table 5 with the cell cryopreservation solution in Table 26 below, the process was the same as in Example 1, and the survival rate of AD-MSCs after freeze-thaw was calculated. These results are shown in Table 27 below.

[0135] Table 26 (Cultivation Experiment) Subsequently, for samples 4–7, the number of viable cells during further culture after freeze-thaw cycles was counted. The cells were washed after freeze-thaw. Next, the cell suspension obtained by resuspending the cells in the culture medium was seeded into 6-well plates (manufactured by Sumitomo Bakelite Corporation, MS-80060) pre-filled with 2 mL of culture medium. The density of the AD-MSCs was adjusted to 5 × 10⁻⁶. 3 ~7.5×10 3 cells / cm 2 The cells were cultured for 3 days. Afterward, the number of viable AD-MSCs was counted. For the cell count, first, the culture supernatant of the 6-well plate was removed, adherent cells were washed once with an appropriate amount of PBS, 500 μL of dissociation agent was added, and the plate was incubated at 37°C for 5 minutes. Then, the mixture containing the dissociation agent was thoroughly stirred, and trypan blue was added. A portion of the mixture was dropped onto a glass slide, and the number of viable AD-MSCs after 3 days of culture was counted using a cell counter. Subsequently, the number of viable cells (N) without amino acid supplementation (sample 4) was counted. i The number of viable cells (N) at different concentrations of alanine and alanine (samples 5-7) j ), calculate the relative viable cell count (N) j / N i ×100(%). These results are shown in Table 28 below.

[0136] Table 27 Table 28 Table 27 shows the survival rate of AD-MSCs after freeze-thaw. Table 28 shows the relative number of viable AD-MSCs during further culture after freeze-thaw. As shown in Table 27, the cell survival rate after freeze-thaw was improved by adding alanine and taurine. Furthermore, as shown in Table 28, the cell survival rate improved in a concentration-dependent manner by setting the alanine concentration to above 0.05 mM. That is, it can be seen that adding amino acids to a sucrose-containing cell cryopreservation solution can improve the cell survival rate after freeze-thaw.

[0137] [Example 8] It was confirmed that using a variety of different cell types in a cell cryopreservation solution containing sucrose and glutathione can improve cell viability during cryopreservation.

[0138] Except for replacing the cell cryopreservation solution in Table 5 with the cell cryopreservation solution in Table 29 below, and replacing the self-made AD-MSCs with adipose-derived stromal cells (commercially available AD-MSCs), umbilical cord-derived mesenchymal cells (UC-MSCs), THP-1 cells, and Jurkat cells, the rest was the same as in Example 1, and the survival rate of AD-MSCs after freeze-thaw was calculated. These results are shown in Table 30 below.

[0139] Table 29 Table 30 Table 30 shows the results of freeze-thaw survival rates of AD-MSCs in various cell species. As shown in Table 30, the cell survival rate after freeze-thaw was improved in all cell species. That is, it can be seen that the cell cryopreservation solution containing sucrose and glutathione can improve the cell survival rate after freeze-thaw even in various cell species.

[0140] [Example 9] It was confirmed that using cell cryopreservation solutions containing different concentrations of ascorbic acid can improve cell viability during cryopreservation.

[0141] Except for replacing the cell cryopreservation solution in Table 5 with the cell cryopreservation solution in Table 31 below, the cryopreservation experiment was conducted in the same manner as in Example 1. In Table 31 below, the number of viable cells (N) without ascorbic acid (Sample 1) is used as the unit of measurement. c Based on the baseline, the number of viable cells (N) at each ascorbic acid concentration (samples 2-7) was determined. d ), calculate the ratio of viable cells after thawing (N) d / Nc ×100(%). These results are shown in Table 31 below.

[0142] Table 31 Table 31 shows the results of the ratio of viable cells after thawing AD-MSCs. As shown in Table 31, compared with the absence of ascorbic acid, the addition of ascorbic acid increased the relative viable cell count after freeze-thaw. In particular, it is evident that setting the ascorbic acid concentration to 1.625 μM or higher can improve cell viability during cryopreservation. In conclusion, the combined use of sucrose and ascorbic acid can further improve cell viability during cryopreservation.

[0143] [Example 10] It was confirmed that different rates of temperature change at freezing points and average cooling rates within the range of 0℃ to -20℃ could improve cell recovery rate after thawing, cell viability during cryopreservation, and the number of live cells after culture.

[0144] (1) Preparation of cell suspension The adipose-derived mesenchymal stem cells (AD-MSCs) cryopreserved as described in (1) were thawed in a 37°C water bath with agitation. The thawed cell culture was transferred to centrifuge tubes (Sumitomo Bakelite Corporation, MS-90150, 15 mL StemFull centrifuge tubes) containing 10 times their volume of HBSS(+). The cells were then centrifuged using a vibrating rotor (400 × g, 5 min) and the supernatant was removed. The recovered AD-MSCs were resuspended in an appropriate amount of culture medium (Solallis Bio Inc., Stem Design™ MSC Culture Medium AF) and seeded into T-150 culture flasks (MS-20600, 150 mL adherent culture flasks, Sumitomo Bakelite) containing culture medium. The AD-MSCs were cultured for 3–4 days to allow them to proliferate to a near-confluence state. After removing the culture supernatant from the AD-MSCs, the adherent cells were washed once with an appropriate amount of PBS (ThermoFisher Scientific, 10010031, pH 7.4, 1000 mL). Subsequently, except for using TrypLE™ (12563029, Select Enzyme (1X), phenol red-free, 500 mL) manufactured by ThermoFisher Scientific as a dissociation agent, the process was the same as in Example 1 to prepare an AD-MSC cell suspension and count the cells.

[0145] (2) Study on cell cryopreservation conditions The addition of HBSS(+) brought the cell density of the cell suspension to 7.0 × 10⁻⁶. 5 ~8.0×10 5 Cells / ml were collected and resuspended. An appropriate amount of the obtained cell suspension was aliquoted into centrifuge tubes and centrifuged using a vibrating rotor (400 × g, 5 min), removing as much supernatant as possible. The precipitate was then resuspended in 2 mL of cell cryopreservation solution to prepare a cell suspension. The cell cryopreservation solution consisted of HBSS(+) containing 10% (w / v) sucrose and 32.5 μM glutathione (same as sample No. 15 in Example 1). 1 mL, 0.5 mL, or 0.25 mL of the cell suspension were aliquoted into 2 mL round-bottom cell cryopreservation tubes (manufactured by Sumitomo Bakelite Co., Ltd., MS-4502), and the tubes were then frozen using any of the methods 1 to 6 described below. For the AD-MSCs, the day after freezing using each method, the tubes were removed from the freezer and 0.5 mL of culture medium preheated to 37°C was added. The tubes were thawed in a 37°C water bath with shaking. Take a portion of the thawed cell suspension, mix it with an equal volume of trypan blue solution, and count the cells using a cell counter. Then, based on the number of viable cells after cryopreservation (N... k ) and the number of live cells before cryopreservation (N) l ), calculate the live cell recovery rate (N) after thawing. k / N l ×100(%). Additionally, the survival rate after freeze-thaw was calculated in the same manner as in Example 1.

[0146] Method 1 (PP (Polypropylene) rack): Place the tube upright in a polypropylene tube rack (AsOne, S500-80G UniRack) at room temperature, and then place the rack in an ultra-low temperature freezer (-80℃) for direct freezing and storage.

[0147] Method 2 (Aluminum rack): Place the tubes upright in a room temperature aluminum block-shaped tube rack (Corning, 423049 CorningCoolRack CF15), and then place the rack in an ultra-low temperature freezer (-80℃) for direct freezing and storage.

[0148] Method 3 (liquid nitrogen gas phase): Place the tube on top of the cryopreservation strip for cell cryopreservation, put it into the gas phase of the liquid nitrogen tank, and freeze it directly.

[0149] Method 4 (PF -0.5℃ / min): Freeze in a programmable freezer with a cooling rate set from 4℃ to -80℃ of -0.5℃ / min, and then transfer to an ultra-low temperature freezer at -80℃.

[0150] Method 5 (PF-1℃ / min): Freeze in a programmable freezer with a cooling rate set to -1.0℃ / min from 4℃ to -80℃, and then transfer to an ultra-low temperature freezer at -80℃.

[0151] Method 6 (PF-10℃ / min): Freeze in a programmable freezer with a cooling rate set to -10.0℃ / min from 4℃ to -80℃, and then transfer to an ultra-low temperature freezer at -80℃.

[0152] (3) Calculation of freezing point and the rate of temperature change when passing through the freezing point The temperature changes of the cell cryopreservation solution in methods 1-6 of Example 10 (2) were measured using cryovials with only the cryopreservation solution added and a thermocouple thermometer (manufactured by TZONE, TC604) (methods 1-3) or a thermometer attached to a programmed cooling system (methods 4-6). Measurements were plotted at 1 second, and cooling curves were drawn based on the measured temperatures. As an example, the cooling curve of the cell cryopreservation solution frozen using method 3 is shown below. Figure 1 .

[0153] Figure 1 This is a graph showing the cooling curve of the cryopreservation solution obtained using method 3. Figure 1 In the diagram, the horizontal axis represents time, and the vertical axis represents temperature. The result is as follows: Figure 1 The cooling curve is shown, and the freezing point is calculated to be -1.1℃ based on the result. Furthermore, the freezing point is as follows... Figure 1 The double-dotted arrow indicates the temperature after latent heat release. Based on this freezing point, the temperature change rate at the freezing point within ±1.0℃ and its corresponding time were calculated linearly using the following formula (1) for each cryopreservation method. In addition, the average cooling rate in the range of 0℃ to -20℃ was also calculated (2) for each method. These results are shown in Table 32 below. Furthermore, the cell recovery rate of AD-MSCs after thawing under each cryopreservation method is as follows: Figure 2 As shown. Furthermore, the survival rates of AD-MSCs after freeze-thaw cycles under various freezing methods are as follows: Figure 3 As shown.

[0154] The rate of temperature change at the freezing point (°C / min) = -2 (°C) / (T) -1 -T +1 )・・・(1) T +1 Time taken to reach the freezing point temperature + 1°C after freezing begins T -1 Time taken to reach the freezing point of -1°C after freezing begins Average cooling rate (°C / min) = -20 (°C) / (T) 20-T0)・・・(2) T0: Time from the start of freezing until the temperature reaches 0°C T 20 Time it takes for the temperature to reach -20°C after freezing begins Table 32 Figure 2 This graph shows the cell recovery rate of AD-MSCs after thawing under various freezing methods. Figure 3 This is a graph showing the survival rate of AD-MSCs after freeze-thaw cycles under various freezing methods. Figure 2 In the graph, the horizontal axis represents the freezing method, and the vertical axis represents the recovery rate. Figure 3 In the graph, the horizontal axis represents the freezing method, and the vertical axis represents the survival rate. For example... Figure 2 and Figure 3 As shown, in any freezing method, both the cell recovery rate after thawing and the cell viability after freeze-thaw are improved. In particular, the cell recovery rate after thawing and the cell viability after freeze-thaw are improved under conditions ranging from PF -0.5℃ / min 500μL to 250μL liquid nitrogen gas phase. That is, it can be seen that by controlling the rate of temperature change through the freezing point within the range of -0.624℃ / min to -58.800℃ / min, the cell recovery rate after thawing and the cell viability after freeze-thaw can be improved. Furthermore, the high-temperature side of the above-mentioned preferred temperature conditions can be achieved by setting the cooling conditions of the programmed cooling device to -0.5℃ / min or -1.0℃ / min, respectively. Additionally, the low-temperature side of the above-mentioned preferred temperature conditions can be achieved by cooling in the gas phase of the liquid nitrogen tank. Furthermore, it can be seen that, based on an average cooling rate within the range of 0 to -20℃, setting it to -0.447 to -22.526℃ / min can improve the cell recovery rate after thawing and the cell viability after freeze-thaw.

[0155] (3) Culture experiment Next, the thawed cell suspension from the study of cell cryopreservation conditions in Example 10 (2) was cultured. In the culture experiment, the 6-well plate was replaced with a T-25 culture flask, and the culture period was changed from 3 days to 2-4 days. The rest of the process was the same as the culture experiment in Example 7. The number of viable AD-MSC cells was counted. The results are as follows: Figure 4 As shown.

[0156] Figure 4 This is a graph showing the number of viable AD-MSCs after culture using various freezing methods. Figure 4 In the graph, the horizontal axis represents the freezing method, and the vertical axis represents the number of viable cells. For example... Figure 4As shown, the number of viable cells after culture was improved in all freezing methods. In particular, the number of viable cells after culture was improved under conditions ranging from PF-1℃ / min 500μL to liquid nitrogen gas phase 250μL. That is, it can be seen that setting the rate of temperature change through the freezing point within the range of -1.000℃ / min to -58.800℃ / min can improve the number of viable cells after culture. Furthermore, it can be seen that, based on an average cooling rate within the range of 0 to -20℃, setting it to -0.736 to -22.526℃ / min can improve the number of viable cells after culture.

[0157] [Example 11] It was confirmed that adding resuscitation fluid during thawing improved the recovery rate of live cells after thawing.

[0158] (Study on thawing conditions for cryopreserved cells) HBSS(+) solution was added to the AD-MSC cell suspension prepared in Example 10 (1) to achieve a cell density of 1.33 × 10⁻⁶ cells / year. 6 Cells / mL. The cell suspension was aliquoted into cryovials at 0.5 mL per vial. The cryovials were placed upright on a polypropylene tube rack that had been pre-cooled overnight in an ultra-low temperature freezer (-80°C) and left to stand overnight to freeze the cells. The obtained cryovials were then removed from the ultra-low temperature freezer and immediately placed in a water bath with shaking (conditions 1-5), or 0.5 mL of thawing medium (Solallis Bio Inc., StemDesign™ MSC Culture Medium AF) preheated to 37°C was added to the cryovials and then shaken in a water bath (conditions 6-10) until the ice on the tube walls melted and the remaining ice in the tube was sufficiently mobile. The cryovials were then placed in the air and inverted at room temperature to mix and allow the ice to melt completely. A portion of the thawed cell suspension was taken and mixed with an equal volume of trypan blue solution, and the cell count was determined using a cell counter. Subsequently, the viable cell recovery rate after thawing was calculated in the same manner as in Example 10 (2). These results are shown in Figure 5 .

[0159] Condition 1: Shake in a 30°C water bath until partially thawed, then invert and mix at room temperature until completely thawed. Condition 2: Shake in a 37°C water bath until partially thawed, then invert and mix at room temperature until completely thawed. Condition 3: Shake in a 40℃ water bath until partially thawed, then invert and mix at room temperature until completely thawed. Condition 4: Shake in a 60℃ water bath until partially thawed, then invert and mix at room temperature until completely thawed. Condition 5: Shake in an 80℃ water bath until partially thawed, then invert and mix at room temperature until completely thawed. Condition 6: After adding 0.5 mL of resuscitation solution, shake in a 30°C water bath until partially thawed, then mix thoroughly at room temperature until completely thawed. Condition 7: After adding 0.5 mL of resuscitation solution, shake in a 37°C water bath until partially thawed, then mix thoroughly at room temperature until completely thawed. Condition 8: After adding 0.5 mL of resuscitation solution, shake in a 40°C water bath until partially thawed, then mix thoroughly at room temperature until completely thawed. Condition 9: After adding 0.5 mL of resuscitation solution, shake in a 60°C water bath until partially thawed, then mix thoroughly at room temperature until completely thawed. Condition 10: After adding 0.5 mL of resuscitation solution, shake in an 80°C water bath until partially thawed, then mix thoroughly at room temperature until completely thawed. Figure 5 This graph shows the viable cell recovery rate of AD-MSCs after thawing using various thawing methods. Figure 5 In the graph, the horizontal axis represents the thawing method, and the vertical axis represents the live cell recovery rate. For example... Figure 5 As shown, adding resuscitation fluid can improve the recovery rate of live cells after thawing.

[0160] [Example 12] It was confirmed that using cell cryopreservation solutions containing surfactants or polyethylene glycol can improve cell viability during cryopreservation.

[0161] Except for replacing the cell cryopreservation solution shown in Table 31 with the cell cryopreservation solutions in Tables 33-44 below, the process was the same as in Example 9. A cryopreservation experiment was performed, and the viable cell ratio after thawing was calculated. Subsequently, except for replacing the cell cryopreservation solution shown in Table 26 with the cell cryopreservation solutions in Tables 33-44 below, the process was the same as in Example 7. A culture experiment was performed, and the relative viable cell count was calculated. These results are shown in Tables 33-44 below. Additionally, in Tables 33-44, the concentrations of surfactant and PEG added are w / v.

[0162] Table 33 —: No test conducted Table 34 —: No test conducted Table 35 —: No test conducted Table 36 —: No test conducted Table 37 —: No test conducted Table 38 —: No test conducted Table 39 Table 40 Table 41 Table 42 —: No test conducted Table 43 —: No test conducted Table 44 Tables 33-44 show the results of the viable cell ratio and relative viable cell count of AD-MSCs after thawing and subsequent freeze-thaw culture. As shown in Tables 33-44, compared with the absence of surfactant, the addition of surfactant improved both the viable cell ratio and the relative viable cell count after thawing. Furthermore, according to the poloxamer results, the higher the proportion of polyethylene oxide (POE) chains in the block copolymer, the higher the relative viable cell count after culture. This is further supported by the fact that the relative viable cell count consistently increased after adding polyethylene glycol (PEG), which acts as a polyethylene oxide. In conclusion, the addition of surfactants and polyethylene glycol, especially polymers containing polyethylene oxide chains, can improve cell viability during freezing and after freeze-thaw culture.

[0163] [Example 13] It was confirmed that using a cell cryopreservation solution containing a reducing agent can improve cell viability during cryopreservation.

[0164] Except for replacing the cell cryopreservation solution shown in Table 26 with the cell cryopreservation solution in Table 45 below, the rest is the same as in Example 7, based on the number of viable cells (N) after culture without the addition of α-lipoic acid (Sample 1). E ) and the number of viable cells (N) after culturing with different concentrations of α-lipoic acid (samples 2-6). f), calculate the relative viable cell count [N] f / N E [×100 (%)]. These results are shown in Table 45 below.

[0165] Table 45 Table 45 shows the relative viable cell count of AD-MSCs after freeze-thaw cycles. As shown in Table 45, the cell viability after freeze-thaw cycles improved in a concentration-dependent manner by adding α-lipoic acid as an antioxidant. In particular, the cell viability was further improved when the concentration of α-lipoic acid was set between 77.55 nM and 9.69 μM. In conclusion, using α-lipoic acid as an antioxidant can improve cell viability after freeze-thaw cycles, and α-lipoic acid can function as an antioxidant in cell cryoprotectants.

[0166] [Example 14] It has been confirmed that exosomes can be cryopreserved using cell cryopreservation solution.

[0167] (1) Preparation of exosomes (extracellular vesicles) Exosomes were prepared from adipose-derived mesenchymal stem cells. Specifically, a proliferation medium (D-MEM / Ham's F-12, manufactured by Fujifilm and Koden Pharmaceuticals) with added cell culture additives (fetal bovine serum) was heated from room temperature to 37°C. Subsequently, adipose-derived mesenchymal stem cells (sample 1 and sample 2) collected from humans were suspended in the proliferation medium and seeded into cell culture flasks. The cell density at seeding was set at 2 × 10⁻⁶ cells / mL. 3 cells / cm 2 Each cell culture flask was placed in an incubator adjusted to 37°C and 5% CO2 and cultured until the cells proliferated to 80-90% confluence. After proliferation, the adipose-derived mesenchymal stem cells were dissociated using trypsin, suspended in serum-free medium (KBM ADSC-4, manufactured by Kohjin-Bio Co., Ltd.), and seeded into new cell culture flasks. The cell density at seeding was 2 × 10⁻⁶ cells / mL relative to the bottom area of ​​the cell culture flask. 4 cells / cm 2 Inoculate the cells using the following method. Place each cell culture flask in an incubator set to 37°C and 5% CO2 for re-culturing, allowing the cells to proliferate to 80-90% confluence.

[0168] Next, after removing the culture medium from each cell culture flask, the cells were washed twice with PBS buffer (with and without PBS buffer). After washing, serum- and animal-derived culture medium (see Patent Nos. 7371975 and 7423016) was added, and the flasks were incubated at 37°C and 5% CO2. After 72 hours, a portion of the culture supernatant (containing the extracellular vesicle composition) was recovered. After culturing for another 3 days, the supernatant was recovered again. The recovered solution was filtered through a 0.22 μm filter and stored at 4°C until the extraction step. Extracellular vesicles were extracted from the filtered solution using a tangential flow filtration device. The extracted sample was filtered through a 100 kDa membrane and concentrated approximately 30-fold. Lactated Ringer's solution was added for washing, and the concentration and washing process was repeated 10 times to obtain the final exosome solution.

[0169] (2) Cryopreservation of exosomes (extracellular vesicles) The exosome solution prepared in Example 14(1) was mixed with cryopreservation compositions (Examples 14-1 and 14-2). Sample 1 was prepared by mixing 1 mL of the cryopreservation composition of Example 14-1 into 1 mL of the exosome solution. Sample 2 was prepared by mixing 0.2 mL of the cryopreservation composition of Example 14-2 into 1 mL of the exosome solution. Control group 1 was prepared by mixing 1 mL of lactated Ringer's solution into 1 mL of the exosome solution. The compositions of Examples 14-1 and 14-2 are shown in Table 46.

[0170] Table 46 All samples were added dropwise to a glass bottle (5 mL) and frozen overnight at -60°C (approximately 16 hours). After freezing, the samples were dried for 20 hours at approximately 5 Pa and approximately -45°C to prepare the lyophilized composition.

[0171] (3) Determination of exosomes 1 mL of water for injection was added to the lyophilized sample to dissolve it. For each dissolved sample, the particle number and particle size were determined using NanoSight (NS300, Malvern) under default conditions. Furthermore, for each sample, the expression levels of CD9 and CD63 in each exosome were determined using a CD9 / CD63 ELISA kit (Cosmo Bio). Control group 2 was performed identically except that the exosome solution prepared in Example 14 (1) was used. The relative expression levels of CD9 and CD63 (100%) relative to control group 2 were then calculated. These results are shown in… Figure 6 and Figure 7 .

[0172] Figure 6 This is a histogram representing the particle distribution. Figure 6 In the diagram, (A) to (D) show the results for control group 1, control group 2, sample 1, and sample 2, respectively. Figure 6 In (A)~(D), the horizontal axis represents particle size, and the vertical axis represents particle concentration. For example... Figure 6 As shown in (A) and (B), compared with the untreated control group 2, control group 1 showed an increase in exosome size due to lyophilization treatment, indicating that lyophilization altered the characteristics of exosomes. In contrast, as... Figure 6 As shown in (C) to (D), compared with the control group 2 which was not freeze-dried, the particle size of samples 1 to 2 hardly changed even after freeze-drying.

[0173] Next, Figure 7 A graph showing the relative expression levels of CD9 / CD63. Figure 7 In the diagram, the horizontal axis represents the sample type, and the vertical axis represents the relative expression levels of CD9 / CD63. For example... Figure 7 As shown, compared with control group 2, the relative expression level of CD9 / CD63 in control group 1 was significantly decreased. In contrast, the decrease in the relative expression level of CD9 / CD63 in samples 1 and 2 was inhibited.

[0174] In summary, the cell cryopreservation composition of the present invention is also suitable for cryopreservation of exosomes as cell-derived components.

[0175] The present invention has been described above with reference to the embodiments and examples, but the present invention is not limited to the above embodiments and examples. Various modifications that can be understood by those skilled in the art can be made to the structure and details of the present invention within the scope of the present invention.

[0176] This application claims priority based on Japanese Patent Application No. 2025-031248 filed on February 28, 2025, Japanese Patent Application No. 2025-079074 filed on May 9, 2025, and Japanese Patent Application No. 2025-145381 filed on September 2, 2025, the entire disclosure of which is incorporated herein by reference.

[0177] The patents, patent applications and documents cited in this specification are used as references to this specification, as if they were specifically described in this specification.

[0178] <Postscript> Some or all of the above-described embodiments and examples may be described in the form of the following notes, but are not limited to the following.

[0179] <Compositions for Cell Cryopreservation> (Note 1) A composition for cell cryopreservation comprising sugars or derivatives thereof, surfactants and / or polymers containing polyoxyethylene chains.

[0180] (Note 2) According to the composition described in Appendix 1, the sugar is a monosaccharide, disaccharide, trisaccharide, and / or polysaccharide.

[0181] (Note 3) According to the composition described in Appendix 2, the disaccharide is selected from sucrose, maltose, sucralose, lactose, cellobiose, lactobionic acid, lactulose, melibiose, and isomaltose.

[0182] (Note 4) The composition according to Appendix 2 or 3, wherein the disaccharide comprises sucrose.

[0183] (Note 5) The composition according to any one of Appendices 2 to 4, wherein the monosaccharide is selected from glucose, galactose, mannose, fructose, xylose, ribose and arabinose.

[0184] (Note 6) The composition according to any one of Appendices 2 to 5, wherein the trisaccharide is selected from raffinose, maltotriose, isomalttriose, panose, and pinotriose.

[0185] (Note 7) The composition according to any one of Appendices 2 to 6, wherein the polysaccharide is selected from dextran, starch, hydroxyethyl starch, hyaluronic acid, glycogen, and alginate.

[0186] (Note 8) The composition according to any one of Appendices 1 to 7, wherein the sugar is a non-reducing sugar and / or a reducing sugar.

[0187] (Note 9) The composition according to any one of Appendices 1 to 8, wherein the sugar derivative is selected from nucleosides, nucleotides, and sugar alcohols.

[0188] (Postscript 10) According to the composition described in Appendix 9, the nucleoside comprises uridine and / or deoxyuridine.

[0189] (Postscript 11) The composition according to Appendix 9 or 10, wherein the sugar alcohol is selected from sorbitol and xylitol.

[0190] (Postscript 12) The composition according to any one of Appendices 1 to 11, wherein the surfactant is a nonionic surfactant.

[0191] (Postscript 13) According to the composition described in Appendix 12, the nonionic surfactant is poloxamer.

[0192] (Postscript 14) The composition according to any one of Appendices 1 to 13, wherein the surfactant comprises polyvinylpyrrolidone (PVP) and / or modified polyalkylene glycol.

[0193] (Postscript 15) According to the composition described in Appendix 13, the modified polyalkylene glycol comprises Soluplus.

[0194] (Postscript 16) The composition according to any one of Appendices 1 to 15, wherein the polymer containing the polyoxyethylene chain comprises poloxamer, polyethylene glycol, polyvinylpyrrolidone (PVP), and / or modified polyalkylene glycol.

[0195] (Postscript 17) The composition according to any one of Appendices 1 to 16 comprises the sugar or a derivative thereof, the surfactant and / or a polymer containing a polyoxyethylene chain.

[0196] (Postscript 18) The composition according to any one of Appendices 1 to 17, It contains the sugar, The concentration of the sugar is 5-25% (w / v).

[0197] (Postscript 19) The composition according to any one of Appendices 1 to 18, The sugars mentioned therein are monosaccharides, disaccharides, and / or trisaccharides. The concentration of the sugar is 5-25% (w / v).

[0198] (Postscript 20) The composition according to any one of Appendices 1 to 19, The sugar derivative therein is a sugar alcohol. The concentration of the sugar alcohol is 1-20% (w / v).

[0199] (Postscript 21) The composition according to any one of Appendices 1 to 20, wherein the concentration of the surfactant is 0.001 to 20% (w / v).

[0200] (Postscript 22) The composition according to any one of Appendices 1 to 21, wherein the concentration of the polymer containing the polyoxyethylene chain is 0.0001 to 20% (w / v).

[0201] (Postscript 23) The composition according to any one of Appendices 1 to 22 further comprises an antioxidant.

[0202] (Postscript 24) According to the composition described in Appendix 23, the antioxidant comprises an antioxidant selected from glutathione, lipoic acid (α-lipoic acid, lipoic acid), ascorbic acid (vitamin C) or derivatives thereof, tocopherol (vitamin E) or derivatives thereof, acetylcysteine, and chelating agents.

[0203] (Postscript 25) According to the composition described in Appendix 24, the ascorbic acid derivative is selected from ascorbic acid-2-glucoside, ascorbic acid-2-phosphate, tetrahexyldecyl ascorbate, and 3-O-ethyl ascorbic acid.

[0204] (Postscript 26) The composition according to Appendix 24 or 25, wherein the tocopherol derivative is selected from α-tocopherol phosphate, tocopherol acetate, and tocopherol succinate.

[0205] (Postscript 27) The composition according to any one of Appendices 24 to 26, wherein the antioxidant comprises alpha-lipoic acid and glutathione.

[0206] (Postscript 28) The composition according to any one of Appendices 23 to 27, wherein the concentration of the antioxidant is 1 nmol / L to 100 mmol / L.

[0207] (Postscript 29) The composition according to any one of Appendices 1 to 28 further comprises amino acids.

[0208] (Note 30) According to the composition described in Appendix 29, the amino acid is selected from alanine, taurine, phenylalanine, methionine, proline, cysteine, serine, lysine, arginine, homoserine, GABA, aminocaproic acid, tryptophan, and glycine.

[0209] (Postscript 31) According to the composition described in Appendix 29 or 30, the concentration of said amino acid is 0.01 to 100 mmol / L.

[0210] (Note 32) The composition according to any one of Appendices 1 to 31 further comprises an aqueous solvent.

[0211] (Postscript 33) According to the composition described in Appendix 32, the aqueous solvent is selected from water, buffer solutions, infusion solutions, and culture media.

[0212] (Postscript 34) According to the composition described in Appendix 33, the buffer solution is selected from Hanks Balanced Salt Solution (HBSS), phosphate buffer, DuPont phosphate buffer, Hepes buffer solution, and amino acid buffer.

[0213] (Postscript 35) The composition according to any one of Appendices 1 to 34, wherein the cell viability after the cell freeze-thaw test is more than 60%.

[0214] (Postscript 36) The composition according to any one of Appendices 1 to 35 is substantially free of organic solvent-based cell cryoprotectants.

[0215] (Postscript 37) According to the composition described in Appendix 36, the organic solvent-based cell cryoprotectant is selected from dimethyl sulfoxide, propylene glycol, ethylene glycol, glycerol, and ethanol.

[0216] (Postscript 38) The composition according to any one of the appendices 1 to 37 is substantially free of animal-derived ingredients.

[0217] (Postscript 39) According to the composition described in Appendix 38, the animal-derived component is a protein, lipid, peptide, hormone, amino acid, and / or vitamin obtained from animal raw materials.

[0218] (Postscript 40) The composition according to any one of Appendices 1 to 39 is substantially free of serum and / or plasma.

[0219] (Postscript 41) The composition according to any one of Appendices 1 to 40 is a chemically defined composition.

[0220] (Postscript 42) The composition according to any one of Appendices 1 to 41, wherein the cells are cell-derived components.

[0221] (Postscript 43) According to the composition described in Appendix 42, the cell-derived component is cell culture supernatant and / or exosomes.

[0222] (Postscript 44) According to the composition described in Appendix 43, the cell culture supernatant and / or exosomes are cell culture supernatants and / or exosomes derived from adipose, bone marrow or umbilical cord mesenchymal stem cells.

[0223] <Cell freezing methods> (Postscript 45) A method for freezing cells, comprising a freezing step of freezing cells in the presence of any one of the compositions described in Appendices 1 to 44.

[0224] (Postscript 46) According to the freezing method described in Appendix 45, in the freezing process, the temperature change rate of the composition as it passes the freezing point is -0.5 to -250°C / minute.

[0225] (Postscript 47) According to the freezing method described in Appendix 45 or 46, in the freezing process, the average cooling rate of the composition in the range of 0°C to -20°C is -0.4 to -50°C / minute.

[0226] (Postscript 48) The freezing method according to any one of Appendices 45 to 47 includes a preservation step of preserving the frozen cells in a frozen state.

[0227] (Postscript 49) The freezing method according to any one of Appendices 45 to 48 includes the step of thawing the frozen cells.

[0228] (Postscript 50) According to any one of the freezing methods in Appendix 45 to 49, the cells are in vitro cultured cells and primary cultured cells, and / or cells isolated from an organism.

[0229] (Postscript 51) According to any one of the freezing methods in Appendix 45 to 50, the cells are selected from mesenchymal cells, immune cells, fibroblasts, epithelioid 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-secreting cells.

[0230] (Postscript 52) The freezing method according to any one of Appendices 45 to 51, wherein the cells are cell-derived components.

[0231] (Postscript 53) According to the freezing method described in Appendix 52, the cell-derived components are cell culture supernatant and / or exosomes.

[0232] (Postscript 54) According to the freezing method described in Appendix 53, the cell culture supernatant and / or exosomes are cell culture supernatants and / or exosomes derived from adipose, bone marrow or umbilical cord mesenchymal stem cells.

[0233] (Postscript 55) The freezing method according to any one of Appendices 45 to 54 is performed in vitro or in vivo.

[0234] <Cell Preparations> (Postscript 56) A cell preparation comprising cells and the composition described in any one of notes 1 to 44.

[0235] (Postscript 57) The cell preparations described in Appendix 56 are in a frozen state or in a solid state.

[0236] (Postscript 58) According to the cell preparations described in Appendix 56 or 57, the cells are selected from 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-secreting cells.

[0237] (Postscript 59) The cell preparation according to any one of Appendices 56 to 58, wherein the cells are cell-derived components.

[0238] (Postscript 60) According to the cell preparations described in Appendix 59, the cell-derived components are cell culture supernatant and / or exosomes.

[0239] (Postscript 61) According to the cell preparations described in Appendix 60, the cell culture supernatant and / or exosomes are cell culture supernatants and / or exosomes derived from adipose, bone marrow or umbilical cord mesenchymal stem cells.

[0240] (Postscript 62) The cell preparation according to any one of Appendices 56 to 61 is obtained by the freezing method described in any one of Appendices 45 to 55.

[0241] (Postscript 63) The cell preparation according to any one of Appendices 56 to 62, wherein the cell preparation is a cryopreserved dried composition of cell preparation.

[0242] <Thawing methods for cell preparations> (Postscript 64) A method for thawing cell preparations, It includes a thawing process for thawing cell preparations in a frozen state. The cell preparation mentioned therein is the cell preparation described in any one of Appendix 56 to 63.

[0243] (Postscript 65) According to the thawing method described in Appendix 64, in the thawing step, the frozen cell preparation is thawed in the presence of cell resuscitation fluid in the cell preparation.

[0244] (Postscript 66) According to the thawing method described in Appendix 65, the thawing solution is added to the frozen cell preparation.

[0245] (Postscript 67) According to the thawing method described in Appendix 65 or 66, the temperature of the thawing fluid is 0–45°C.

[0246] (Postscript 68) The thawing method according to any one of Appendices 65 to 67, wherein the thawing solution is an aqueous solvent.

[0247] (Postscript 69) According to the thawing method described in Appendix 68, the aqueous solvent is selected from water, buffer solution, infusion solution and culture medium.

[0248] (Postscript 70) The thawing method according to any one of Appendices 64 to 69, wherein the cell preparation is thawed at a temperature of 20 to 80°C.

[0249] <Disposal Methods> (Postscript 71) A treatment method includes an administration step of administering the cell preparation described in any one of Appendices 56 to 62 to a subject.

[0250] (Postscript 72) According to the treatment method described in Appendix 71, the subject is a disease patient who can be treated with the cells contained in the cell preparation.

[0251] Industrial availability As described above, according to the present invention, for example, cells can be protected during cell freezing even without substantially adding organic solvent-based cell cryoprotectants. Therefore, the present invention can be considered to have extremely high application value in fields such as cell therapy and regenerative medicine.

Claims

1. A composition for cell cryopreservation comprising sugars or derivatives thereof.

2. The 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 sucrose, maltose, sucralose, lactose, cellobiose, lactobionic acid, lactulose, melibiose, and isomaltose.

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

5. The composition according to any one of claims 1 to 4, further comprising an antioxidant.

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

7. The composition according to claim 5 or 6, wherein the antioxidant comprises lipoic acid and glutathione.

8. The composition according to any one of claims 5 to 7, wherein the concentration of the antioxidant is 1 nmol / L to 100 mmol / L.

9. The composition according to claim 1 or 8, further comprising an aqueous solvent.

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

11. The composition according to any one of claims 1 to 10, wherein the cell viability after the cell freeze-thaw test is more than 60%.

12. The composition according to claim 1 or 11 is substantially free of organic solvent-based cell cryoprotectants.

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

14. A method for freezing cells, comprising a freezing step of freezing cells in the presence of the composition according to any one of claims 1 to 13.

15. A cell preparation comprising cells and the composition according to any one of claims 1 to 13.

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