Cell preparation

Abstract

To provide a cell preparation in which a cell is protected from cryopreservation, and self-replication performance and a function of the cell is maintained even after freezing / thawing.SOLUTION: A cell preparation includes: a polymer having the viscosity-average molecular weight larger than 3,000 and 500,000 or less, which is a polymer including monomers having a hydrophilic group as a repeating unit or a salt thereof; saccharide having the viscosity-average molecular weight of 3,000 or less or a salt thereof; a cell; and a pharmaceutically acceptable carrier.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a cell preparation. [Background technology]

[0002] With the rapid development of regenerative medicine research in recent years, regenerative medicine such as cell therapy is being actively conducted. In such regenerative medicine and regenerative medicine research, regenerative medicine products, which are cell and tissue processed products derived from cells such as pluripotent stem cells (embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), somatic stem cells, and somatic cells, are used.

[0003] Cell and tissue processed products contain living target cells and show medicinal effects due to the diverse characteristics of these target cells. There is a demand for a stable supply of cells with the same standards in terms of efficacy and safety.

[0004] The target cells are usually cultured in large quantities, and then cryopreserved for use, optionally through a process such as formulation. However, cryopreserved cell lines may result in heterogeneity or insufficient survival rate after thawing. This is known to be because, in the cell cryopreservation mechanism, ice crystals grow inside the cells during the freezing and / or thawing process, causing damage to the cell membrane and intracellular structure, or denaturation of the cells' proteins, resulting in fatal damage to the cells. Therefore, when cryopreserving cells, it is important to prevent intracellular freezing, and low molecular weight compounds such as dimethyl sulfoxide (DMSO), glycerin, and propylene glycol are usually used for cryopreservation of cells by adding them to buffer solutions such as culture media as intracellular permeation type cryoprotective reagents (Patent Document 1). Of these, DMSO is the most commonly used, and has a good effect on protecting cells and organelles. However, DMSO has high permeability to cells (Non-Patent Document 1), and there is concern about the effects on the human body if DMSO is used as a cryoprotective reagent and used as it is as a physiological carrier solution for cell preparations.

[0005] Therefore, as described in Patent Document 2, a method for preparing a cell preparation has also been proposed, which comprises mixing thrombin serum produced by a specified method with a platelet-rich plasma (PRP) composition or a platelet concentrate composition, and further mixing with one or more of a cell extract, a cell composition, tricalcium phosphate (TCP), a bone substitute, chitosan, hyaluronic acid, a cream, a cream mask, adipose cells, adipose tissue, bone marrow concentrate, lubricin, cd-gelatin, botulinum toxin, calcium gluconate, and / or stem cells. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 63-216476 [Patent Document 2] JP 2019-206594 A [Non-patent literature]

[0007] [Non-Patent Document 1] REJUVENATION RESEARCH Volume 18 Number 5 (2015): 422-436, Mary Ann Liebert, Inc. Summary of the Invention [Problem to be solved by the invention]

[0008] However, the cell preparation of Patent Document 2 had a low cell viability when cryopreserved, making it impractical.

[0009] The present invention has been made in consideration of the above problems, and provides a cell preparation in which cells are protected during cryopreservation from freezing to thawing, the cells exhibit a high cell survival rate after thawing, and the quality of the cells after thawing is appropriately maintained. In particular, the present invention aims to provide a cell preparation in which cells are protected in the frozen state and against thawing without basically using chemical substances such as dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), etc., and without basically adding serum or serum-derived proteins. [Means for solving the problem]

[0010] The present invention relates to a cell preparation comprising a polymer having a viscosity average molecular weight of more than 3000 and not more than 500,000, the polymer containing a monomer having a hydrophilic group as a repeating unit, or a salt thereof, a saccharide having a viscosity average molecular weight of not more than 3000, or a salt thereof, cells, and a pharma- ceutically acceptable carrier. The salt of the polymer or saccharide is preferably a metal salt, a halogen salt, or a sulfate salt. The metal salt is preferably a salt of an alkali metal or an alkaline earth metal. The alkali metal or alkaline earth metal may be selected from sodium, potassium, calcium, or the like. The halogen may be chlorine, bromine, or the like.

[0011] As used herein, a pharma- ceutically acceptable carrier refers to a solid or liquid for maintaining cells in a viable state, the osmotic pressure and pH of which are adjusted to a composition and concentration that allow cells to survive.

[0012] The pharma- ceutically acceptable carrier is preferably at least one selected from a buffer, a stabilizer, and an isotonicity agent.

[0013] In the present invention, it is desirable that the cell preparation contains, as a main component, a polymer having a viscosity average molecular weight of more than 3000 and not more than 500,000, the polymer containing a monomer having a hydrophilic group as a repeating unit, or a salt thereof, and, as a secondary component, a saccharide having a viscosity average molecular weight of not more than 3000, or a salt thereof. In this specification, the main component refers to the component having the highest weight ratio among the components dissolved in the cell preparation. Components dissolved in the cell preparation other than the main component are secondary components.

[0014] In the polymer used in the present invention, the hydrophilic groups contained in the repeating unit of the monomer having a hydrophilic group are preferably not modified, or if modified, the number of the hydrophilic groups is less than 50% of the total number. It is presumed that the hydrophilic groups in the polymer are involved in the protection of cells in the cell preparation, the vitrification of the solvent, and the replacement of the sugar with a molecular weight of 3000 or less with the water around the cells, and if the hydrophilic groups are modified to be hydrophobic, the cryopreservation effect on the cells is reduced. Therefore, it is preferable to exclude hydrophobic polymers such as carboxypolyamino acids as the main component.

[0015] In addition, the cell preparation of the present invention preferably does not contain dimethyl sulfoxide, which is an intracellularly penetrating cryoprotectant, and preferably does not contain cytotoxic cryoprotectants such as propylene glycol and ethylene glycol, which are harmful to cells after thawing.

[0016] In the present invention, the viscosity average molecular weight of the polymer or its salt is desirably 400,000 or less, particularly 200,000 or less, because the viscosity can be adjusted to a low level and the preparation is easy to handle as a cell preparation.

[0017] Preferably, the pharma- ceutically acceptable carrier of the present invention includes an isotonicity agent and a buffer.

[0018] It is preferable that the cell preparation of the present invention further contains an amino acid.

[0019] It is preferable that the cell preparation of the present invention further contains a monosaccharide. The monosaccharide referred to here is different from the saccharide referred to in "saccharide or a salt thereof having a viscosity-average molecular weight of 3000 or less."

[0020] In the cell preparation, the monomer having a hydrophilic group of the polymer of the present invention is preferably a monomer having a hydrophilic group which is at least one selected from the group consisting of a hydroxyl group and a carboxylic acid group and a salt thereof.

[0021] A preferred cell preparation is one in which the polymer further contains, as a repeating unit, a nitrogen-containing monomer having an optionally substituted amino group or an optionally substituted amide group.

[0022] The cell preparation is preferably a polymer that is an alternating copolymer of the monomer having a hydrophilic group and the nitrogen-containing monomer.

[0023] A preferred cell preparation is one in which the monomer having a hydrophilic group is a monomer having a hydroxyl group substituted at an equatorial position.

[0024] A cell preparation containing a polymer having a viscosity average molecular weight of 5,000 or more or a salt thereof is preferred.

[0025] Preferred are cell preparations in which the macromolecule is a macromolecule that comprises multiple sugar residues.

[0026] Preferred are cell preparations in which the saccharide is a monosaccharide, disaccharide, or oligosaccharide.

[0027] Preferred are cell preparations in which the sugar is glucose, fructose, galactose, or uronic acids in which the alcohol group of these sugars is oxidized, or amino sugars in which the alcohol group is substituted with an amino group, sucrose, cleavage products of glycosaminoglycans, constituent monosaccharides of glycosaminoglycans, or polymers or combinations thereof.

[0028] Preferred are cell preparations in which the saccharide is glucuronic acid or N-acetylglucosamine.

[0029] The isotonicity agent in the cell preparation is preferably an inorganic salt, such as a potassium salt, a sodium salt, a calcium salt, D-sorbitol, or D-mannitol.

[0030] Preferred are cell preparations in which the inorganic salt is a chloride salt.

[0031] Preferred are cell preparations in which the buffer is a phosphate or bicarbonate.

[0032] A preferred cell preparation is one in which the amino acid is proline.

[0033] A cell preparation in which the monosaccharide is glucose is preferred.

[0034] Preferred are cell preparations which further comprise a proteinaceous active agent.

[0035] Preferred are cell preparations in which the protein active agent is a magnesium salt or a calcium salt.

[0036] The concentration of the polymer or a salt thereof in the cell preparation is preferably 5 w / v % or more and 20 w / v % or less.

[0037] The concentration of the sugar or its salt in the cell preparation is preferably 0.1 w / v % or more and 10 w / v % or less.

[0038] The cell preparation of the present invention may be a cell preparation in the form of a cell sheet or a sheet-like cell culture.

[0039] The cell preparation of the present invention may be in a frozen state.

[0040] Preferably, the frozen cell preparation comprises a plurality of cryopreserved cells, the cells having at least 80% viability at the time of freezing and recovered following thawing.

[0041] The cell preparation of the present invention is preferably a cell preparation in which the cell in the cell preparation is a stem cell or a cell derived from a stem cell.The cell may also be a gene-edited cell, and the editing method may be a commonly used method such as adeno-associated virus vector or CRISPR Cas9.

[0042] The cells are stem cells, mesenchymal stem cells, Muse cells, ES cells, iPS cells, hair papilla cells, hair follicle stem cells, sebaceous gland stem cells, neural stem cells, olfactory ensheathing cells, bone marrow mesenchymal cells, dental pulp cells, Schwann cells, neural progenitor cells, dopaminergic neural progenitor cells, corneal stromal cells, corneal endothelial cells, corneal epithelial cells, retinal pigment epithelial cells, myoblasts, cardiac myocytes, nephron progenitor cells, differentiated kidney cells, kidney stem cells, hepatocytes, hepatic progenitor cells, colon epithelial stem cells, colon absorptive system epithelial cells The cell preparation is preferably a cell preparation selected from the group consisting of cells, colon secretory epithelial cells, DFAT cells, vascular endothelial cells, vascular pericytes, smooth muscle cells, bone marrow mononuclear cells, chondroblasts, cultured cartilage, epidermal cells, epidermal stem cells, skin fibroblasts, dental pulp (stem) cells, ameloblasts, periodontal ligament stem cells, osteoblasts, osteoclasts, dendritic cells (activated), mononuclear cells, T cells, CAR-T cells, γδT cells, NK cells, CAR-NK cells, hematopoietic stem cells, platelets, plasma, and red blood cells. Although platelets, plasma, and red blood cells are generally classified as cellular components, they are defined as cells in the present specification.

[0043] The cell preparation is preferably a corneal epithelial sheet, a retinal pigment sheet, a myocardial cell sheet, a hepatic cell sheet, an epidermis sheet, a cartilage sheet or a periosteum sheet, or a stem cell sheet such as a mesenchymal stem cell sheet.

[0044] The cell preparation is preferably for the treatment and / or prevention of nerve disorders, skin defects, cerebrovascular disorders, spinal cord injuries, brain trauma, central nervous system disorders, Parkinson's disease, corneal disorders, Stevens-Johnson syndrome, ocular pemphigoid, corneal chemical injuries / burns, cicatricial keratoconjunctival epitheliopathy, bullous keratopathy, infectious keratitis, corneal dystrophy, retinal optic neuropathy, corneal epithelial stem cell deficiency, liver disorders, hyperammonemia, ischemic heart disease, heart failure, thrombocytopenia, arteriosclerosis obliterans, burns, wounds, cartilage damage, complications during bone marrow transplantation, aplastic anemia, metabolic liver diseases, colitis, arteriosclerosis obliterans, ischemia, peripheral arterial disease, non-arteriosclerosis, bone system diseases, osteoarthritis, bone tumors, osteonecrosis, lower limb ischemic disease, cancer, rheumatism, osteoporosis, or muscular dystrophy.

[0045] The cell preparation is preferably a cell preparation administered intrabone marrow, intravenously, intramuscularly, intraperitoneally, intraocularly, or intracerebrally.

[0046] The present invention also relates to a treatment method comprising the step of administering a therapeutically effective amount of the cell preparation of the present invention to a patient suffering from a disease selected from the group consisting of nerve disorders, skin defects, cerebrovascular disorders, spinal cord injuries, brain trauma, central nervous system disorders, Parkinson's disease, corneal disorders, Stevens-Johnson syndrome, ocular pemphigoid, corneal chemical injuries / burns, cicatricial keratoconjunctival epitheliopathy, bullous keratopathy, infectious keratitis, corneal dystrophy, retinal optic neuropathy, corneal epithelial stem cell deficiency, liver disorders, hyperammonemia, ischemic heart disease, heart failure, thrombocytopenia, arteriosclerosis obliterans, burns, wounds, cartilage damage, complications during bone marrow transplantation, aplastic anemia, metabolic liver diseases, colitis, arteriosclerosis obliterans, ischemia, peripheral arterial disease, non-arteriosclerosis, bone system diseases, osteoarthritis, bone tumors, osteonecrosis, lower limb ischemic diseases, cancer, rheumatism, osteoporosis, and muscular dystrophy.

[0047] The present invention also relates to a solution for preserving cells, comprising a polymer or a salt thereof having a viscosity average molecular weight of more than 3,000 and not more than 500,000, the polymer including a monomer having a hydrophilic group as a repeating unit, a saccharide or a salt thereof having a viscosity average molecular weight of 3,000 or less, and a pharma- ceutically acceptable carrier.

[0048] It is preferable that the solution for preserving cells of the present invention further contains an amino acid.

[0049] It is preferable that the solution for preserving cells of the present invention further contains a monosaccharide.

[0050] The present invention also relates to use of a solution comprising a polymer or a salt thereof having a viscosity average molecular weight of more than 3,000 and not more than 500,000, the polymer including a monomer having a hydrophilic group as a repeating unit, a saccharide or a salt thereof having a viscosity average molecular weight of 3,000 or less, and a pharma- ceutically acceptable carrier, for preserving cells.

[0051] The cells are stem cells, mesenchymal stem cells, Muse cells, ES cells, iPS cells, hair papilla cells, hair follicle stem cells, sebaceous gland stem cells, neural stem cells, olfactory nerve sheathing cells, bone marrow mesenchymal cells, dental pulp cells, Schwann cells, neural progenitor cells, dopaminergic neural progenitor cells, corneal stromal cells, corneal endothelial cells, corneal epithelial cells, retinal pigment epithelial cells, myoblasts, cardiac myocytes, nephron progenitor cells, differentiated kidney cells, kidney stem cells, hepatic cells, hepatic progenitor cells, colonic epithelial stem cells, colonic absorptive system epithelium The preferred uses are cells, colon secretory epithelial cells, DFAT cells, vascular endothelial cells, vascular pericytes, smooth muscle cells, bone marrow mononuclear cells, chondroblasts, cultured cartilage, epidermal cells, epidermal stem cells, skin fibroblasts, dental pulp (stem) cells, ameloblasts, periodontal ligament stem cells, osteoblasts, osteoclasts, dendritic cells (activated), mononuclear cells, T cells, CAR-T cells, γδT cells, NK cells, CAR-NK cells, hematopoietic stem cells, platelets, plasma, or red blood cells.

[0052] According to the present invention, cell preparations can be safely preserved without changing the properties of the cells, even without using cytotoxic compounds such as DMSO or ethylene glycol, which are intracellularly penetrating cryoprotectants.

[0053] In addition, since the cell preparation of the present invention does not require serum or serum-derived proteins, it is not contaminated with bacteria or viruses, and there are no problems such as differences between lots. It is possible to add proteins that are not contaminated with bacteria or viruses. It is also possible to include chemical substances that are cytotoxic at low concentrations that do not impair the function of the cells in the cell preparation.

[0054] The "viscosity average molecular weight" of the polymer or saccharide used in the present invention is determined by the following method and formula.

[0055] Intrinsic viscosity measurement: (1) Dissolve a specified amount of NaCl in ion-exchanged water at 30°C to prepare a 0.2 M NaCl solution (standard solution). (2) Dissolve a polymer or sugar sample in a standard solution at 30°C to prepare a stock solution. Measure the viscosity of each of the standard solution and the stock solution, and adjust the relative viscosity of the stock solution to 2.0 to 2.4 with respect to the standard solution. (3) Dilute the 30°C stock solution 5 / 4, 5 / 3, and 5 / 2 times with the 30°C standard solution. (4) Measure the viscosity of the standard solution, undiluted solution, and diluted solution at 30°C. Use an E-type viscometer to measure the viscosity. (5) The viscosity of the original solution and the diluted solution divided by the viscosity of the standard solution is the relative viscosity (η r ) and derive the reduced viscosity based on the following formula: TIFF2024177228000001.tif2146Here, η sp : Reduced viscosity of polymer or sugar [mL / g], η r : relative viscosity of polymer or sugar [-], C: concentration of polymer or sugar [g / mL]. (6) Plot the relationship between the concentration of the polymer or sugar and the reduced viscosity of the polymer or sugar, and draw an approximate straight line. The intercept of the approximate straight line (where the concentration of the polymer or sugar = 0) is the limiting viscosity.

[0056] Viscosity average molecular weight: The viscosity average molecular weight is calculated from the intrinsic viscosity. TIFF2024177228000002.tif3559The viscosity average molecular weight M is calculated from the above Mark Hoying-Sakurada equation using the intrinsic viscosity derived from measurement and the values ​​of K and α published in literature, etc.

[0057] K and α are values ​​that vary depending on the type of polymer. The values ​​of K and α are disclosed in many published documents, such as the "Polymer Materials Handbook" (compiled by the Society of Polymer Science). The viscosity average molecular weight can be calculated using these published values.

[0058] For example, in the case of hyaluronic acid, K = 3.6 × 10 -4 and α = 0.78, and for pullulan and gelatin, K = 9 × 10 -4 , α=0.5, and for dextran, K=6.3×10 -8 , α = 1.4, and K = 5.8 × 10 for chondroitin sulfate. -4 , α=0.74.

[0059] In the case of monosaccharides, disaccharides, and compounds that are considered to be monomolecular, the molecular weight is clearly specified from the structural formula, and therefore in the present invention, the molecular weight specified from the structural formula is treated as a fictitious viscosity average molecular weight. Effect of the Invention

[0060] In the cell preparation of the present invention, the formation of ice crystals in the solvent portion of the cell preparation is prevented in the frozen state by the polymer component having a viscosity average molecular weight of more than 3000 and not more than 500,000 contained in the preparation, and the cell preparation is vitrified. This can suppress the bursting of cells in the cell preparation due to the formation of ice crystals. Furthermore, since the cell preparation of the present invention contains low molecular weight sugars having a viscosity average molecular weight of not more than 3000, water molecules near the boundary tissue between the cell and the solvent are replaced with sugar molecules, and as a result, the formation and growth of ice crystals near the cell membrane can also be inhibited. This can also suppress cell membrane damage to cells in the frozen state. In the cell preparation of the present invention, a high cryopreservation effect is obtained by the intracellular non-permeable cryopreservation agent, which has not been achieved in the past, and therefore damage to and changes in quality of living cells in the cell preparation can be prevented during the freezing and thawing process of the cell preparation. Therefore, according to the cell preparation of the present invention, the cell preparation can be stored safely and securely.

[0061] Furthermore, since the cell preparation of the present invention does not contain intracellularly penetrating chemicals such as DMSO or ethylene glycol, nor serum or serum-derived proteins, the cell preparation can be directly administered after freezing and thawing.

[0062] The cell preparation of the present invention can be stably stored for a long period of time at -27°C or lower, i.e., in a deep freezer, etc. The range of the frozen storage temperature is not limited as long as it is -27°C or lower, but the upper limit is desirably -70°C or lower, preferably -80°C or lower. The lower limit is desirably -196°C or higher, preferably -150°C or higher. [Brief description of the drawings]

[0063] [Figure 1] FIG. 13 is a graph showing the cell viability of cells after cryopreservation and thawing of the cell preparation of the present invention containing RPE cells. [Diagram 2] FIG. 1 shows images of cells cultured after cryopreservation and thawing of the cell preparation of the present invention containing RPE cells. [Diagram 3]FIG. 1 shows the cell viability after cryopreservation and thawing of the cell preparation of the present invention containing normal canine mesenchymal stem cells derived from canine bone marrow. [Figure 4] FIG. 1 shows images of cells cultured after cryopreservation and thawing of the cell preparation of the present invention containing normal canine mesenchymal stem cells derived from canine bone marrow. [Figure 5A] FIG. 1 shows the differentiation of cells into bone cells after cryopreservation and thawing of the cell preparation of the present invention containing normal canine mesenchymal stem cells derived from canine bone marrow. [Figure 5B] FIG. 1 shows the differentiation of cells into adipocytes after cryopreservation and thawing of the cell preparation of the present invention containing normal canine mesenchymal stem cells derived from canine bone marrow. [Figure 5C] FIG. 1 shows the differentiation of cells into chondrocytes after cryopreservation and thawing of the cell preparation of the present invention containing normal canine mesenchymal stem cells derived from canine bone marrow. [Figure 6A] FIG. 1 shows the amount of HGF produced by cells after cryopreservation and thawing of the cell preparation of the present invention containing normal canine mesenchymal stem cells derived from canine bone marrow. [Figure 6B] FIG. 1 shows the amount of IL-10 produced by cells after cryopreservation and thawing of the cell preparation of the present invention containing normal canine mesenchymal stem cells derived from canine bone marrow. [Figure 7] FIG. 1 shows an analysis of CD antigen expression in cells after cryopreservation and thawing of the cell preparation of the present invention containing normal canine mesenchymal stem cells derived from canine adipose tissue. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0064] The cell preparation of the present invention comprises a polymer or a salt thereof having a viscosity average molecular weight of more than 3000 and not more than 500,000, a saccharide or a salt thereof having a viscosity average molecular weight of not more than 3000, cells, and a pharma- ceutically acceptable carrier. The polymer contained in the cell preparation of the present invention is a polymer containing a monomer having a hydrophilic group as a repeating unit. The cell preparation of the present invention is a cell preparation that contains a monomer having a hydrophilic group as a repeating unit and comprises a mixture of a polymer or a salt thereof having a viscosity average molecular weight of more than 3000 and not more than 500,000, and a saccharide or a salt thereof having a viscosity average molecular weight of not more than 3000 as a cryopreservation agent.

[0065] The viscosity average molecular weight of the polymer or its salt used in the present invention is desirably 400,000 or less, particularly 200,000 or less, because the viscosity can be adjusted to a low level and the preparation is easy to handle as a cell preparation.

[0066] The "viscosity average molecular weight" of the polymer or saccharide used in the present invention means a value calculated by the following method and formula.

[0067] The method for measuring the intrinsic viscosity and the method for calculating the viscosity average molecular weight using the intrinsic viscosity are described below. Intrinsic viscosity measurement: (1) Dissolve a specified amount of NaCl in ion-exchanged water at 30°C to prepare a 0.2 M NaCl solution (standard solution). (2) Prepare a stock solution by dissolving the polymer or sugar sample in a standard solution at 30°C. If the polymer or sugar sample is obtained in the form of a solution, the solid portion obtained by removing the solvent from the solution is used as the polymer or sugar sample. In the case of a mixed sample of polymers and sugars, a mixed sample containing multiple polymers, or a mixed sample containing multiple sugars, each substance is separated and fractionated, and the solvent is removed from each substance to obtain the polymer or sugar sample. If the polymer and / or sugar are unknown, the substance is identified for the polymer and / or sugar by HPLC, LC-MS, LC-IR, etc. If multiple unknown polymers and / or sugars are included, each component is separated and fractionated, and the substance for each polymer and / or sugar is identified by HPLC, LC-MS, LC-IR, etc., and the viscosity average molecular weight is calculated as described below. If the mixture contains impurities in the polymer or sugar, but the viscosity is not affected (for example, impurities such as metal salts), the mixture is used as the polymer or sugar sample. If the solution contains impurities that affect the calculation of the viscosity average molecular weight, remove the impurities or separate the polymers and sugars before measuring. Measure the viscosity of the standard solution and the stock solution, and adjust the relative viscosity of the stock solution to 2.0 to 2.4 compared to the standard solution. (3) Dilute the 30°C stock solution 5 / 4, 5 / 3, and 5 / 2 times with the 30°C standard solution. (4) Measure the viscosity of the standard solution, undiluted solution, and diluted solution at 30°C. Use an E-type viscometer to measure the viscosity. (5) The viscosity of the original solution and the diluted solution divided by the viscosity of the standard solution is the relative viscosity (η r ) and derive the reduced viscosity based on the following formula: TIFF2024177228000003.tif2146Here, η sp : Reduced viscosity of polymer or sugar [mL / g], η r : relative viscosity of polymer or sugar [-], C: concentration of polymer or sugar [g / mL]. (6) Plot the relationship between the concentration of the polymer or sugar and the reduced viscosity of the polymer or sugar, and draw an approximate straight line. The intercept of the approximate straight line (where the concentration of the polymer or sugar = 0) is the limiting viscosity. Viscosity average molecular weight: The viscosity average molecular weight is calculated from the intrinsic viscosity. TIFF2024177228000004.tif3559The viscosity average molecular weight M is calculated from the intrinsic viscosity obtained by measurement and the values ​​of K and α published in the literature, etc., in the Mark Hoying-Sakurada formula above. For example, in the case of hyaluronic acid, K = 3.6 × 10 -4 and α = 0.78 to calculate the viscosity average molecular weight M.

[0068] K and α are values ​​that vary depending on the type of polymer. The values ​​of K and α are disclosed in many published documents, such as the "Polymer Materials Handbook" (compiled by the Society of Polymer Science). The viscosity average molecular weight is calculated using these published values.

[0069] In the present invention, the molecular weight calculated by this method is regarded as the viscosity average molecular weight. In the case of monosaccharides and disaccharides such as sucrose and glucuronic acid, and compounds considered to be monomolecular, the molecular weight is clearly specified from the structural formula, so the molecular weight specified from the structural formula is treated as the viscosity average molecular weight.

[0070] According to the cell preparation of the present invention, the cell preparation can be cryopreserved without decreasing the cell viability while appropriately maintaining the cell properties. The cell preparation of the present invention includes a polymer having a predetermined molecular weight and a large number of hydrophilic groups, and this polymer forms a polymer chain containing hydrophilic groups during the cooling process to freeze, trapping the water molecules of the solvent in the cell preparation within the polymer chain matrix. By restricting the molecular motion of the trapped water molecules, when the cell preparation is cooled, the water can be solidified and / or frozen in a vitrified state without crystallization. Usually, a freezing method called vitrification is a method in which, when a solution freezes, a solute (a cryoprotectant for preventing freeze damage) is excluded from the crystals, increasing the salt concentration in the remaining solution, and an osmotic pressure difference occurs between the inside and outside of the cell, dehydrating the inside of the cell and vitrifying the inside of the cell, and is applied to cells with a particularly low survival rate after thawing. In such a method, in order to make water easier to vitrify, the concentration of the solute (cryoprotectant) is increased or the cooling rate is increased. However, it has been known that increasing the osmotic pressure difference increases damage to the cells, and that recrystallization occurs during thawing, causing damage to the cells. There are also technical difficulties involved.

[0071] According to the cell preparation of the present invention, the inside of the cells is dehydrated and vitrified by the action of the polymer chains formed by the polymers contained in the cell preparation, thereby suppressing the formation of ice crystals within the cells and further reducing the osmotic shock in the cells during freezing, which is a problem with conventional vitrification methods.

[0072] Furthermore, the viscosity average molecular weight of the polymer is more than 3000 and 500000 or less, so that the amorphous glass state of the cell preparation in the frozen state is stabilized. Therefore, even if the cell preparation is frozen, the cells in the cell preparation are not easily damaged, and the cells can be stably and efficiently cryopreserved. Therefore, the survival rate of the thawed cells after thawing the frozen and stored cell preparation of the present invention is maintained high. If the viscosity average molecular weight of the polymer is 3000 or less, vitrification may not occur well. If the viscosity average molecular weight of the polymer is more than 500000, the viscosity increases significantly, and the solubility decreases, and foaming occurs during preparation, which may cause problems such as poor handling. For example, the viscosity average molecular weight of the polymer in the cell preparation of the present invention is preferably 5000 or more. Also, the viscosity average molecular weight is preferably 400000 or less, or even 200000 or less, and particularly preferably 150000 or less. It is considered that the viscosity can be adjusted to a low level, and the cell preparation is easy to handle.

[0073] The polymer contained in the cell preparation of the present invention is a polymer containing a monomer having a hydrophilic group as a repeating unit. The hydrophilic group is, for example, a hydroxyl group and a carboxylic acid group and a salt thereof. The polymer contained in the cell preparation of the present invention may also contain a nitrogen-containing monomer having an amino group which may be substituted or an amide group which may be substituted as a repeating unit. Furthermore, the polymer contained in the cell preparation of the present invention preferably has a hydroxyl group at an equatorial position in its structure. This is believed to allow the water of the solvent to be more effectively trapped in the matrix formed by the polymer chains during freezing.

[0074] The monomer having a hydrophilic group is, for example, a sugar residue. In this case, the polymer contained in the cell preparation of the present invention may be a polymer containing a repeating unit of sugar residues bound by glycosidic bonds and derivatives thereof. Examples of sugar residues include monosaccharides, or monosaccharides in which the hydroxyl and / or hydroxymethyl groups of monosaccharides are substituted, for example, monosaccharides in which the hydroxyl and / or hydroxymethyl groups are substituted with at least one substituent selected from the group consisting of a carboxyl group, an amino group, an N-acetylamino group, a sulfoxy group, a methoxycarbonyl group, and a carboxymethyl group, but are not limited thereto.

[0075] Examples of monosaccharides include triose, tetrose, pentose, hexose, and heptose. For example, examples of pentoses include ribose, arabinose, xylose, lyxose, xylulose, ribulose, and deoxyribose. Examples of hexoses include glucose, mannose, galactose, fructose, sorbose, tagatose, fucose, fuculose, and rhamnose.

[0076] For example, the monosaccharide substituted with a carboxyl group includes uronic acid. Examples of the uronic acid include glucuronic acid, iduronic acid, mannuronic acid, and galacturonic acid. Examples of the monosaccharide substituted with an amino group include amino sugars. Examples of the amino sugar include glucosamine, galactosamine, mannosamine, and muramic acid. Examples of the monosaccharide substituted with an N-acetylamino group include N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, and N-acetylmuramic acid. Examples of the monosaccharide substituted with a sulfooxy group include galactose-3-sulfate. Examples of the monosaccharide having multiple substituents include N-acetylglucosamine-4-sulfate, iduronic acid-2-sulfate, glucuronic acid-2-sulfate, N-acetylgalactosamine-4-sulfate, neuraminic acid, and N-acetylneuraminic acid.

[0077] For example, the polymer used in the cell preparation of the present invention is a polymer containing monosaccharides as repeating units as exemplified above. For example, the polymer contained in the cell preparation of the present invention may be a polymer containing pentose, hexose, or uronic acid, which may be substituted, or a combination thereof, as repeating units. The polymer contained in the cell preparation of the present invention may also be an alternating copolymer of a monomer having a hydrophilic group and a nitrogen-containing monomer. The nitrogen-containing monomer may be, for example, an amino sugar. In this case, for example, the polymer contained in the cell preparation of the present invention may be a glycosaminoglycan. It may also be a sulfated polysaccharide in which one or more hydroxyl groups are substituted with sulfooxy groups. Examples of the polymer contained in the cell preparation of the present invention include, but are not limited to, hyaluronic acid, dextran, pullulan, and chondroitin sulfate.

[0078] The polymer used in the cell preparation of the present invention may be naturally derived or may be chemically synthesized. Commercially available polymers may be used as they are. A naturally derived polymeric compound or a commercially available polymeric compound having a larger molecular weight may be used, and the cleavage product may be obtained by subjecting the compound to hydrolysis, enzyme treatment, subcritical treatment, or the like, and the molecular weight may be adjusted to prepare the polymer for the cell preparation of the present invention. In addition, each monomer may be naturally derived, may be a naturally derived monomer that has been modified or substituted, or may be chemically synthesized. For example, preferably, the monomer contained in the polymer for the cell preparation of the present invention is a biological component. It is considered that the polymer itself is unlikely to have an adverse effect on the cells in the cell preparation, and the cell preparation can be directly administered.

[0079] The hydrophilic groups of the polymer used in the cell preparation of the present invention are preferably not modified, or modified but not exceeding 50% of the total number of hydrophilic groups, i.e., no substituents are introduced into the polymer chain, or even if they are introduced, the hydrophilic groups are preferably not exceeding 50% of the total number of hydrophilic groups. It is presumed that the hydrophilic groups of the polymer, particularly the OH group, NH2 group, and COOH group, contribute to cell protection, vitrification of the solvent, and replacement of sugar with water around the cells, and therefore it is advantageous for improving the survival rate of the cells if these functional groups are not modified. Furthermore, there is a risk that the reagents used to modify the hydrophilic groups of the polymer may adversely affect the cryoprotective effect of the polymer, or even that the cell preparation may not be usable as a cell / tissue processed product for regenerative medicine purposes.

[0080] In addition, it is believed that the hydrophilic group of the polymer can hold low molecular weight sugars by hydrogen bonding, and it is presumed that the presence of the polymer holding low molecular weight sugars around the cells in the cell preparation can promote the replacement of water molecules and sugars near the cell membrane. Therefore, if the hydrophilic group is modified, the retention effect of the hydrophilic group for low molecular weight sugars is reduced, and even if low molecular weight sugars coexist, they cannot fully contribute to improving the survival rate of cells. From this point of view, it is not preferable to modify, for example, OH groups or NH2 groups with carboxylic acids.

[0081] The salt of the polymer or saccharide contained in the cell preparation of the present invention may be a metal salt, a halogen salt, or a sulfate salt of the polysaccharide. As the metal salt, an alkali metal or an alkaline earth metal salt is preferable. As the alkali metal or the alkaline earth metal, sodium, potassium, calcium, etc. are selected. As the halogen, chlorine, bromine, etc. can be used. It is considered that the salt lowers the freezing point of the solvent, thereby contributing to the vitrification of the solvent.

[0082] The cell preparation of the present invention contains a saccharide or its salt having a viscosity average molecular weight of 3000 or less. When the cell preparation contains a saccharide having such a small molecular weight together with a polymer, the water molecules near the cell membrane of the cell in the cell preparation are replaced by the saccharide, and the formation and growth of ice crystals near the cell membrane are suppressed, and as a result, cell membrane damage can be significantly suppressed. That is, the saccharide or its salt used in the cell preparation of the present invention can function as a component for cell protection. The saccharide contained in the cell preparation of the present invention can be, for example, a monosaccharide, disaccharide, or oligosaccharide having a molecular weight of 3000 or less, preferably 2000 or less, more preferably 1000 or less.

[0083] The saccharide may be, for example, a monosaccharide exemplified above as a monomer constituting a polymer. For example, the saccharide may be glucose, fructose, galactose, or uronic acid in which the alcohol group of these is oxidized, or an amino sugar in which the alcohol group is substituted with an amino group, sucrose, trehalose, or a polymer or combination thereof. The saccharide may also be, for example, a fragment of a polymer used for the cell preparation of the present invention, such as hyaluronic acid, dextran, pullulan, or chondroitin sulfate. Although not particularly limited as long as it does not impair the effect of the present invention, the saccharide may be, for example, a cleavage product (fragment) of glycosaminoglycan, that is, a monosaccharide constituting glycosaminoglycan, a disaccharide thereof, or an oligosaccharide thereof.

[0084] Preferably, saccharide is the cleavage product of hyaluronic acid.Therefore, preferably, the saccharide used for the cell preparation of the present invention is glucuronic acid or N-acetylglucosamine, or the disaccharide or oligosaccharide thereof.Preferably, saccharide can be glucuronic acid or its modified compound, or the disaccharide or oligosaccharide thereof.

[0085] The term "cleavage product" as used in the present invention means a compound having a smaller molecular weight than the original polymer, which is considered to be obtained when a polymer is subjected to hydrolysis, enzyme treatment, subcritical treatment, or the like. The polymer used in the cell preparation of the present invention may be a polymer having a viscosity average molecular weight of more than 3000 and not more than 500,000 obtained by treatment of a larger polymer compound as described above, and the saccharide used in the cell preparation of the present invention may be a saccharide having a viscosity average molecular weight of not more than 3000 obtained by treatment of the polymer used in the cell preparation of the present invention. The cleavage product may be a monomer that is a component of the original polymer and / or a polymer of various polymerization degrees of the monomer and / or a mixture thereof.

[0086] "Subcritical processing" means contacting the raw material to be extracted with a subcritical fluid as an extracting solvent that has been brought into a subcritical state under conditions of a predetermined temperature and a predetermined pressure. For example, when water is raised to a pressure of 22.12 MPa or more and a temperature of 374.15°C or more, it is in a state that is neither liquid nor gas. This point is called the critical point of water, and hot water with a temperature and pressure close to the critical point is called subcritical water. Using the hydrolysis action of this subcritical water, desired components can be obtained from the raw material to be extracted. Conditions for subcritical processing in the present invention include, for example, a temperature of 150°C or more and 350°C or less, and the subcritical processing pressure can be equal to or higher than the saturated vapor pressure at each temperature, but can be, for example, 0.5 MPa or more and 25 MPa or less. After the subcritical processing, components with a molecular weight of less than a predetermined value are separated and collected, and can be used as the cleavage product in the present invention. In addition, there are no particular limitations on the hydrolysis or enzyme processing, and reagents and processing methods that are commonly used can be used without problems.

[0087] The polymer and saccharides having a predetermined molecular weight in the present invention may be obtained simultaneously by a single subcritical treatment. That is, the polymer and saccharides in the cell preparation of the present invention may be a subcritical treatment product of a polymer compound having a first molecular weight distribution in a molecular weight range of more than 3000 and not more than 500000 as a viscosity average molecular weight, and a second molecular weight distribution in a molecular weight range of not more than 3000 as a viscosity average molecular weight. Thus, for example, the method for producing the cell preparation of the present invention may include a step of obtaining the polymer and saccharides for the cell preparation of the present invention by dissolving a polymer, which is a polysaccharide having a molecular weight of more than 500000 as a viscosity average molecular weight, in water and then performing an extraction treatment under subcritical conditions of water. However, the polymer and saccharides for the cell preparation of the present invention may be a combination of those treated and / or separated based on molecular weight in separate treatment steps.

[0088] The cell preparation of the present invention contains the polymer or its salt at a concentration of about 1 w / v% or more and 50 w / v% or less. If the concentration is lower than 5 w / v%, the solvent portion of the cell preparation may not be vitrified well. If the concentration is higher than 20 w / v%, the viscosity may become too high, which may deteriorate the handling properties of the cell preparation. For example, the concentration of the polymer or its salt is preferably 5 w / v% or more, and more preferably 10 w / v% or more. Also, the concentration of the polymer or its salt is preferably 50 w / v% or less, and more preferably 20 w / v% or less. Preferably, the concentration of the polymer or its salt is 5 w / v% or more and 20 w / v% or less.

[0089] The concentration of the saccharide or its salt in the cell preparation of the present invention is about 0.1 w / v% or more and about 10 w / v% or less. That is, the ratio of the polymer to the saccharide in the present invention is preferably about 10:1. If the concentration of the saccharide or its salt is less than 0.1 w / v%, the effect of the present invention may not be sufficiently obtained. On the other hand, even if the saccharide is added to a concentration of 10 w / v% or more, it is difficult to obtain further effect as a cell protective component.

[0090] The cells of the cell preparation of the present invention are not particularly limited. The polymers and saccharides having a predetermined molecular weight, which can function as cryoprotectants in the cell preparation of the present invention, are intracellularly non-permeable cryoprotectants, and therefore can provide cryoprotective action with high survival rates while maintaining their functionality for various cells. Therefore, the cell preparation of the present invention can be prepared using various cells, and then stored after performance tests, etc. Therefore, the cell preparation of the present invention can be provided immediately when needed. Cell preparations for various cells, including the polymers and saccharides having a predetermined molecular weight of the present invention, can be formulated, well cryopreserved, and administered after thawing for use in various cell therapies. It is believed that the use of the cell preparation of the present invention can stably supply cells of the same standard in terms of efficacy and safety at low cost while ensuring safety. In the cell preparation of the present invention, ice crystal formation and recrystallization are effectively suppressed during freezing and thawing of the cells, and therefore the cell preparation of the present invention is particularly suitable as a cell preparation for, for example, mammalian cells having a complex structure.

[0091] The cell preparation of the present invention can be used as a regenerative medicine product. Therefore, preferably, but not limited to, the cells of the cell preparation of the present invention are, for example, pluripotent stem cells such as embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells), or stem cells such as somatic stem cells, tissue stem cells, and adult stem cells, or cells derived from stem cells. According to the cell preparation of the present invention, stem cells can be formulated while maintaining the properties of stem cells. Therefore, even stem cells for regenerative medicine use can be formulated without the risk of differentiation, and can be used as a regenerative medicine product.

[0092] The cells of the cell preparation of the present invention may be cells collected from a living body, or cells or cell lines obtained by culturing these cells outside the body may be used. It may be preferable to use cells of the same lot obtained by expansion culture, which are divided into small portions, in that the same action and effect can be stably obtained. The cells may be cells that have been repeatedly frozen and thawed as appropriate. The origin of the cells is not particularly limited. They may be cells of heterologous or homologous origin. The cell preparation of the present invention may be prepared using cells of any animal species. For example, the cells may be derived from any organism to which the cell preparation is administered. Examples of such organisms include humans, non-human primates, dogs, cats, pigs, horses, goats, sheep, rodents, rabbits, etc. The cells of the cell preparation of the present invention may be autologous cells (also called autologous cells) or non-autologous cells (also called allogeneic cells). Therefore, the cell preparation of the present invention may be formulated as an off-the-shelf cell preparation. Specific examples of cells will be described later, but the cells of the cell preparation of the present invention are not limited to these.

[0093] The cells contained in the cell preparation of the present invention may be, for example, mesenchymal stem cells obtained from various tissues. For example, mesenchymal stem cells derived from bone marrow, umbilical cord, umbilical cord blood, endometrium, placenta, amniotic membrane, chorion, decidua, dermis, dental follicle, periodontal ligament, dental pulp, tooth germ, adipose tissue, blood vessel (periphery), skeletal muscle, or synovium may be included. The cells of the present invention may also be hematopoietic stem cells, neural stem cells, hepatic stem cells, skeletal muscle stem cells, epithelial stem cells, epidermal stem cells, retinal stem cells, or adipose stem cells.

[0094] Furthermore, the cells contained in the cell preparation of the present invention may be, for example, Muse cells separated from mesenchymal stem cells as cells with differentiation potential, cells edited by reprogramming, genome editing, activation, etc. from somatic stem cells or mature cells collected from a living body, such as iHep cells induced to become hepatocytes by introducing a gene into fibroblasts, or Clip cells reprogrammed into hepatic progenitor cells capable of proliferation by adding a low molecular weight compound to mature hepatocytes.

[0095] The cell types of the cell preparation of the present invention may be cells derived from various tissues. For example, cells derived from tissues such as hair, nerve, cornea, retina, muscle, heart, kidney, liver, stomach, intestine, blood vessels, cartilage, skin, or teeth are preferably used.

[0096] For example, examples of suitable cells in the cell preparation of the present invention include hair papilla cells, hair follicle stem cells, sebaceous gland stem cells, neural stem cells, olfactory nerve sheathing cells, bone marrow mesenchymal cells, dental pulp cells, Schwann cells, neural progenitor cells, dopaminergic neural progenitor cells, corneal stromal cells, corneal endothelial cells, corneal epithelial cells, retinal pigment epithelial cells, retinal ganglion cells, myoblasts, nephron progenitor cells, differentiated kidney cells, kidney stem cells, hepatic cells, hepatic progenitor cells, colon epithelial stem cells, colon absorptive epithelial cells, colon secretory epithelial cells, mature adipocytes, DFAT cells, vascular endothelial cells, vascular pericytes, capillary stem cells, cardiac myocytes, cardiac myoblasts, Examples of such cells include smooth muscle cells, peripheral blood mononuclear cells, bone marrow mononuclear cells, skin fibroblasts, chondroblasts, chondrocytes, cultured chondrocytes, cultured cartilage, epidermal stem cells, epidermal cells, dermal stem cells, dental pulp (stem) cells, periodontal ligament stem cells, dental follicle stem cells, dental papilla stem cells, odontoblasts, ameloblasts, osteoblasts, osteocytes, skeletal myoblasts, osteoclasts, dendritic cells (activated), mononuclear cells, T cells such as CAR-T cells (chimeric antigen receptor T cells) and γδT cells, NK cells such as CAR-NK cells, hematopoietic stem cells, platelets, plasma, red blood cells, activated lymphocytes, and Langerhans cells.

[0097] The cells contained in the cell preparation of the present invention may be in the form of a cell sheet or a sheet-shaped cell culture that has been cultured to form a sheet-shaped tissue. Examples of the cells that constitute the cell sheet or the sheet-shaped cell culture include the cells exemplified above, and examples of the cell sheet include a myoblast cell sheet, a cardiac cell sheet, a corneal epithelial sheet, a corneal endothelial sheet, a retinal pigment sheet, a retina sheet, a hepatic cell sheet, a cultured skin sheet, a cultured epidermal sheet, a cartilage sheet, a periosteum sheet, or a stem cell sheet. The cell sheet may be a composite cell sheet having a two-layer structure of the cells exemplified above and supporting cells.

[0098] The cell preparation of the present invention is useful for treating various diseases, and can be used, for example, in regenerative medicine for repairing organs damaged by disease or injury, cell replacement therapy, cardiovascular regenerative medicine, cancer immunotherapy and cell therapy, neural regeneration therapy, and T cell infusion therapy, for example, cell infusion therapy of cells optionally introduced with genes. Diseases that can be treated with the cell preparation of the present invention include, but are not limited to, for example, nerve disorders, skin defects, epidermolytic ichthyosis, cerebrovascular disorders including cerebral infarction, cerebellar diseases, hypoxic-ischemic encephalopathy, spinal cord injury, brain trauma, central nervous system diseases, Parkinson's disease, corneal disorders, Stevens-Johnson syndrome, ocular pemphigoid, corneal chemical injuries and burns, cicatricial keratoconjunctival epitheliopathy, bullous keratopathy, infectious keratitis, corneal dystrophies such as granular corneal dystrophy, retinal optic nerve disorders such as age-related macular degeneration, retinitis pigmentosa, and glaucoma, corneal epithelial stem cell deficiency, periodontitis, liver disorders, hyperammonemia, ischemic heart disease, idiopathic dilated cardiomyopathy, heart failure, thrombocytopenia, arteriosclerosis obliterans, burns, and wounds. , cartilage damage, complications during bone marrow transplantation, aplastic anemia graft-versus-host disease, intractable viral infections, renal failure, metabolic liver disease, colitis, arteriosclerosis obliterans, ischemia, peripheral arterial disease, non-arteriosclerosis, bone system disease, osteoarthritis, bone tumor, osteonecrosis, lower limb ischemic disease, cancer, rheumatism, osteoporosis, or muscular dystrophy, and can be used, for example, for transplantation into a site of myocardial damage, myocardial regeneration, bone formation, cartilage formation, formation of new blood vessels, white matter reconstruction, middle ear tympanic membrane regeneration, nerve regeneration therapy, promotion of nerve axon elongation, regeneration of peripheral myelinated nerves and central nerves, visual function regeneration, functional regeneration of neural circuits, regeneration of hair follicles and epidermis, or regeneration of tissues around hair follicles, regeneration of mucosa in intestinal diseases, creation of metabolic organs, etc. Thus, the cell preparation of the present invention is a cell preparation for treating and / or preventing the diseases exemplified above, or for regenerating cells or tissues.

[0099] The cell preparation of the present invention can be produced by dissolving a polymer and a sugar in a pharma- ceutically acceptable carrier, such as a buffer solution, and suspending the above-mentioned cells. In this case, the preparation can be appropriately carried out using a conventional method. The formulation can be in the form of a liquid such as a solution or suspension, a powder obtained by lyophilizing the liquid, or a solid such as a tablet obtained by molding the powder.

[0100] The pharma- ceutically acceptable carrier used in the present invention refers to a solid or liquid for maintaining cells in a viable state, and the osmotic pressure and pH are adjusted to a composition and concentration that allow cells to survive.

[0101] The pharma- ceutically acceptable carrier is preferably at least one selected from a buffer, a stabilizer, and an isotonicity agent.

[0102] For example, the pharma- ceutically acceptable carrier of the present invention may be a buffer solution containing a buffering agent. The buffer solution may be, for example, an aqueous pH-buffered salt solution, and the pH is adjusted to be within a predetermined pH range. Such a buffer solution may function to minimize the change in pH in the cell preparation.

[0103] Preferably, the pharma- ceutically acceptable carrier of the present invention includes an isotonicity agent and a buffer.

[0104] The buffer that can be used in the present invention adjusts the pH of the cell preparation of the present invention to a predetermined pH range. The pH range of the cell preparation is not particularly limited as long as it is within a medicamentarily, pharmacologically, and / or physiologically acceptable range, but for example, the pH of the cell preparation of the present invention is about pH 5.5 to 8.0, preferably about pH 6.0 to 7.6, and more preferably about pH 6.5 to 7.5. However, the preferred pH range of the cell preparation may vary depending on the target cell type of the cell preparation. In addition, for example, when the hydrophilic group of the monomer having the hydrophilic group is a carboxylic acid group, an aqueous solution containing a polymer obtained by polymerizing such a monomer may be acidic. In addition, when the above-mentioned saccharide has a carboxylic acid group, the aqueous solution may be acidic due to such saccharide. In such a case, the pH of the cell preparation can be easily adjusted to an appropriate range by the buffer. And, when necessary, the cell preparation is adjusted to a pH range more suitable for cell survival by using a material that is usually used for adjusting the pH of an aqueous solution. Additionally, the cells are combined with the pharma- ceutically acceptable carrier before and / or after adjusting the pH of the pharma- ceutically acceptable carrier.

[0105] Examples of buffers include bicarbonate buffers such as sodium bicarbonate, potassium dihydrogen phosphate, and disodium hydrogen phosphate. In addition, PBS (phosphate buffered saline), Ringer's solution, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), HBSS (Hanks' Balanced Salt Solution), MOPS (3-(N-morpholino)propanesulfonic acid), TES (N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid), TRIS (tris(hydroxymethyl)aminomethane), TEST (a combination of TES / TRIS), and the like may also be used.

[0106] The tonicity agent of the present invention can be an inorganic salt or a sugar alcohol, and the inorganic salt can be at least one selected from sodium salt, sodium salt, or calcium salt, and the sugar alcohol can be at least one selected from sorbitol or mannitol. The sodium salt can be sodium chloride. The sodium chloride can be physiologically active saline.

[0107] The osmotic pressure of the pharma- ceutically acceptable carrier of the present invention can be appropriately set within a range acceptable to the living body depending on the cell type of the cell preparation, the state of the cells, etc., and can be adjusted to about 200 to 1000 mOSM / L. When the osmotic pressure is within this range, in addition to the cell protective effect of the polymer and sugars, dehydration of the cells is promoted and vitrification inside the cells proceeds more satisfactorily when the cell preparation of the present invention is frozen.

[0108] The ionic strength of the cell preparation of the present invention is about 0.05 to 0.5. The cell preparation of the present invention may contain amino acids, sugars, inorganic salts and / or vitamins, and examples of inorganic salts include chlorides such as sodium chloride, potassium chloride, calcium chloride, and mixtures thereof. The content of inorganic salts in the cell preparation of the present invention is, for example, 0.15 to 15 mg / mL, and preferably 0.4 to 10 mg / mL.

[0109] Stabilizers that may be used in the present invention include polyethylene glycol and the like.

[0110] The cell preparation of the present invention may contain other components that are useful for cell culture or freezing. For example, the cell preparation of the present invention may further contain a cryoprotective support material that assists the cryoprotective action of the cryopreservation agent of the present invention as another component. The cryoprotective support material is a material different from the material employed as a saccharide or salt thereof having a viscosity average molecular weight of 3000 or less. Examples of such materials include amino acids that are known to form ice nuclei in a solution at a temperature higher than the temperature at which intracellular water freezes. Examples of such amino acids include glycine, alanine, valine, asparagine, isoleucine, glutamine, proline, and histidine. Of these, proline is more preferred. In addition, the cryoprotective support material may be a cell membrane non-permeable cryoprotectant, and in this case, examples include saccharides and dextran. Examples of saccharides include dextrose, mannose, galactose, fructose, raffinose, lactose, sucrose, maltose, glucose, sorbitol, mannitol, and trehalose. Of these, dextrose is more preferred.

[0111] The cell preparation of the present invention may contain one or more of the above cryoprotective support substances. The content of the above cryoprotective support substance in the cell preparation of the present invention is not particularly limited, but may be about 1 / 10 of the total concentration of the polymer or its salt and the saccharide or its salt, which are the cryopreservation agent of the present invention. For example, the concentration of the above cryoprotective support substance is about 0.1 w / v% or more and 10 w / v% or less. If the concentration of the above cryoprotective support substance is 10 w / v% or more, the effect of the cryopreservation agent of the present invention is difficult to obtain, and the survival of the cells in the cell preparation after freezing and thawing may be reduced. If the concentration of the above cryoprotective support substance is 10 w / v% or less, the effect of the cryoprotective support substance is difficult to obtain.

[0112] The cell preparation of the present invention may also contain a protein activator as another component. Examples of such protein activators include magnesium chloride, magnesium sulfate, calcium chloride, and mixtures thereof. These substances have the function of activating intracellular enzymes.

[0113] The cell preparation of the present invention is preferably an aqueous solution. When preparing an aqueous solution, it is preferable to use distilled water, distilled water for injection, distilled water for cell culture, distilled water for embryo manipulation, or the like as a solvent.

[0114] In addition to the cells, the polymers and saccharides, and other components exemplified above, the cell preparation of the present invention may further contain various optional components, such as components that enhance the viability, engraftment and / or function of cells, and other active ingredients useful for treating target diseases, within the scope of the present invention. Here, "optional components" refers to components that may or may not be included. As such optional components, known ones can be used, and examples thereof include components other than active ingredients that can be included in general medicines, quasi-drugs, etc. In addition, the pharmaceutically acceptable carrier of the present invention may contain a basal medium. Examples of such basal media include Dulbecco's modified Eagle's Medium (D-MEM) medium, Eagle's Minimum Essential Medium (E-MEM) medium, αMEM medium, RPMI 1640 medium, Medium 199 medium, IMDM medium, MCDB201 medium, Fischer's medium, F-12 medium, and mixed media thereof.

[0115] The cell preparation of the present invention, which contains the polymer and saccharide as a cryopreservation agent, has low cytotoxicity to cells, and does not require an increased concentration of solutes to restrict the molecular motion of water in the solvent during freezing, so that the cell in the cell preparation is less damaged even when cryopreserved. In addition, unlike conventional vitrification methods, the cell preparation of the present invention does not require a high cooling rate to reduce osmotic shock during cooling for cryopreservation. Therefore, the cell preparation of the present invention can be cryopreserved efficiently in a simple manner without reducing the survival rate of the cells. Then, it can be thawed as needed and then administered to a subject. Since the toxicity of the cryopreservation agent to cells is also low, the cells in the cell preparation after thawing maintain their functions well and retain the same properties as before freezing. Therefore, it is also possible to culture the thawed cells as needed before the next cryopreservation, and re-formulate them to prepare a cell preparation.

[0116] The inventors have clarified that when an aqueous solution containing a polymer having a viscosity average molecular weight of more than 3000 and not more than 500000, which contains a monomer having a hydrophilic group as a repeating unit, and a saccharide having a viscosity average molecular weight of not more than 3000 is thermally analyzed by differential scanning calorimetry (DSC), the aqueous solution has a glass transition temperature of about -23°C ± 4°C. Furthermore, in an aqueous solution containing the polymer and saccharide of the present invention, not only is ice crystal formation suppressed during the cooling process, but recrystallization of water during the subsequent heating process, i.e., during melting, is also suppressed. From this, it is considered that when a cell preparation containing the polymer and saccharide of the present invention is frozen and stored, the glass state of the solvent of the cell preparation is stabilized in the frozen state. Since the glass state is further stabilized in this way, the cell preparation of the present invention shows a high cell survival rate after freezing and thawing without the need for the addition of protein components such as serum derived from human or bovine or serum-derived components (e.g., albumin), which have been conventionally required. Therefore, the cell preparation of the present invention is a chemically defined cell preparation that is free from the risk of infectious diseases and is not affected by differences between lots. The cryopreservation agent of the present invention, which is composed of a polymer and a saccharide, has low cytotoxicity, so that in the cell preparation of the present invention, cells that have been cryopreserved and then thawed exhibit a high survival rate. Of course, it is possible to add a protein that is free from the risk of infectious diseases to the cell preparation.

[0117] As described above, the cell preparation of the present invention does not require a large cooling rate for cell protection during cooling. Therefore, after preparation, the cell preparation of the present invention is cooled to −27° C. or lower, which is the glass transition temperature of the aqueous solution containing the polymer of the present invention and the sugar of the present invention. For example, the cell preparation of the present invention is placed in a freezing treatment container or the like and left in a deep freezer at −80° C., and the cells in the cell preparation can be frozen and stored stably without cell damage. A low temperature such as −150° C., which is usually required for cryopreservation of cells, is not necessarily required. Therefore, it is possible to eliminate the need for a special container or preparation of liquid nitrogen for cryopreservation of the cell preparation in liquid nitrogen. The operation related to the cryopreservation of the cell preparation can be significantly simplified. For example, the cell preparation of the present invention can be cooled at a cooling rate of about 10° C. / min or less. Preferably, the cooling rate is about 1° C. / min or less. It is considered that at this level of cooling rate, the inside of the cells is appropriately dehydrated, the vitrification of the intracellular fluid occurs well, and a high cell cryoprotection effect can be obtained. There is no need for rapid operation for rapid cooling required in conventional vitrification methods. Therefore, operability is significantly improved. Furthermore, in the cell preparation of the present invention, recrystallization of water during thawing is also suppressed. Therefore, not only freezing and storage, but also a series of steps up to thawing of the cell preparation can be easily and efficiently performed without the need for special procedures. The range of cryopreservation temperature of the cell preparation is not limited to -27°C or lower. Of course, the cell preparation of the present invention may be cryopreserved in the gas phase above liquid nitrogen at temperatures from about -150°C or lower to -180°C or lower. From the viewpoint of operation, the range of cryopreservation temperature is desirably -70°C or lower, preferably -80°C or lower. The lower limit is desirably -196°C or higher, preferably -150°C or higher.

[0118] The storage period of the frozen cell preparation of the present invention is not particularly limited, so long as the frozen cells retain the same properties after thawing as before freezing, but may be, for example, 1 week or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 1 year or more, or more.

[0119] The polymer of the present invention, which has a viscosity average molecular weight of more than 3000 and not more than 500000, and which contains a monomer having a hydrophilic group as a repeating unit, is a non-permeable cryoprotective reagent, and is therefore considered to have low cytotoxicity. In addition, in the cell preparation of the present invention, the sugars used together with the polymer function for cell protection, so that the properties of the cells do not change during cryopreservation of the cell preparation. Therefore, the cell preparation of the present invention can be cryopreserved while maintaining the properties of the cells at the same level as before freezing.

[0120] Thus, in the cell preparation of the present invention, the vitrified state of the solvent is stabilized in the frozen state, and the toxicity is low, so that the cells can be stably stored for a long period of time. In this specification, long-term stable storage means, for example, that the viability of the thawed cells after thawing the frozen and stored cell preparation of the present invention is reduced by less than 10%, preferably less than 5%, after 5 months, or by less than 20%, preferably less than 10%, after 6 months, or by less than 15%, preferably less than 30%, after 12 months, based on the viability of the cells immediately before storage. In addition, in this specification, long-term stable storage means, for example, that when the cell preparation is frozen and stored at -80°C for a long period of time, thawed, and then stored at 4°C, even 24 hours after thawing, the viability is reduced by less than 5%, based on the viability of the cells immediately after thawing. In the frozen cell preparation of the present invention, the cells are considered to be cryopreserved under less stressful conditions than when DMSO or the like is contained as a cryoprotectant. Therefore, a very high cell viability can be obtained for the cells after thawing of the cell preparation of the present invention. Furthermore, not only the cells immediately after thawing but also the cells refrigerated after thawing can exhibit high cell viability. According to the cell preparation of the present invention, cells can be stably cryopreserved for a long period of time without changing the properties of the cells.

[0121] The polymer of the present invention has a viscosity average molecular weight of more than 3000 and less than 500000, and the polymer contains a monomer having a hydrophilic group as a repeating unit, and is preferably, for example, a polymer that is a biological component. By using such a polymer or its salt, a cryopreserved cell preparation can be thawed, and the thawed cells can be administered as is. For example, a preferred polymer of the present invention is hyaluronic acid. In particular, it is hyaluronic acid with a viscosity average molecular weight of 400000 or less, preferably 200000 or less. More particularly, it is hyaluronic acid with a viscosity average molecular weight of more than 3000, more preferably more than 5000, and 60000 or less, more preferably 20000 or less.

[0122] The present invention also relates to a method for treating a disease in a subject, comprising administering an effective amount of the cell preparation of the present invention to the subject in need thereof. Thus, the cell preparation of the present invention may be a cell preparation or disease therapeutic agent containing a therapeutically effective amount of the cells as exemplified above. The diseases to be treated include those exemplified above for the cell preparation of the present invention.

[0123] The present invention also relates to a method for regenerating cells or tissue comprising administering an effective amount of the cell preparation of the present invention, which may comprise the step of administering cells of the cell preparation of the present invention to a tissue of an animal in need thereof.

[0124] Furthermore, the present invention further relates to a treatment method comprising the step of administering a therapeutically effective amount of the cell preparation of the present invention to a patient suffering from the above-mentioned disease.

[0125] The administration method of the cell preparation of the present invention is typically, but not limited to, direct application to tissue. The administration route may be, for example, intravascular administration, particularly intravenous administration, or intramuscular administration, intrathecal administration, intraperitoneal administration, intestinal administration, rectal administration, vaginal administration, intraocular administration, intracerebral administration, subcutaneous administration, nasal administration, sublingual administration, oral administration, inhalation, transdermal administration, implantation, spraying on the surface of an organ, and direct (local) administration to the affected area by applying a sheet or the like. The administration may be, for example, by injecting cells with a syringe, inserting with a catheter, or surgically transplanting cells. In addition, for example, when the cell preparation of the present invention is in the form of a cell sheet or the like, when applying to tissue, the cell preparation may be fixed to the target tissue by a fastening means such as a suture or staple. Intravenous administration may be preferred from the viewpoint of reducing the burden on the subject. In addition, in order to reduce the burden on the subject and the practitioner, it may be desirable to use an injection.

[0126] The dosage of the cell preparation of the present invention may vary depending on the type of disease, the severity of symptoms, the condition of the subject (e.g., patient) (e.g., weight, age, symptoms, physical condition, etc.), and dosage form, but is preferably an amount sufficient to obtain a sufficient therapeutic effect while suppressing the occurrence of side effects. For example, the dosage is 1x10 cell number. 3 ~1x10 12 Pieces / time, preferably 1x10 4 ~1x10 11 The frequency of administration is typically once per treatment, but if the desired effect is not obtained, it is possible to administer multiple times. That is, the above-mentioned dose may be administered multiple times as a single dose, or this dose may be administered in multiple doses. It may also be administered continuously. The cell preparation of the present invention may also be administered together with one or more other drugs. Alternatively, the cell preparation of the present invention may not be administered directly, but may be used to obtain a therapeutic composition for administration to a subject by dispersing the cell preparation containing the cells as it is, or the cells separated by a method such as centrifugation, in an infusion solution such as physiological saline, glucose solution, Ringer's solution, lactate Ringer's solution, or acetate Ringer's solution.

[0127] Furthermore, the present invention relates to a solution for preserving cells as exemplified above, which is contained in the cell preparation of the present invention, and which comprises a polymer having a viscosity average molecular weight of more than 3000 and not more than 500,000, which contains a monomer having a hydrophilic group as a repeating unit, or a salt thereof, a saccharide having a viscosity average molecular weight of not more than 3000, or a salt thereof, and a pharma- ceutically acceptable carrier. According to the solution for preserving cells of the present invention, ice crystal formation and recrystallization during freezing and thawing of the cells to be preserved can be effectively suppressed with a simple operation, and the cells can be stably preserved. Cells can be suitably preserved in the solution for preserving cells of the present invention while maintaining the properties and viability of the cells.

[0128] The solution for preserving cells of the present invention may further contain an amino acid. Such an amino acid may act as a cryoprotective support material to assist the cryoprotective action of the solution for preserving cells of the present invention. Examples of such amino acids include glycine, alanine, valine, asparagine, isoleucine, glutamine, proline, and histidine. Among these, proline is more preferred.

[0129] The solution for preserving cells of the present invention may further contain a monosaccharide. Such a monosaccharide can act as a cell membrane-impermeable cryoprotectant. An example of the monosaccharide is glucose.

[0130] The present invention further relates to use of a solution comprising a polymer or a salt thereof having a viscosity average molecular weight of more than 3,000 and not more than 500,000, the polymer including a monomer having a hydrophilic group as a repeating unit, a saccharide or a salt thereof having a viscosity average molecular weight of 3,000 or less, and a pharma- ceutically acceptable carrier, for preserving cells.

[0131] Cells that can be suitably preserved by the solution for preserving cells of the present invention include the cells exemplified above for the cell preparation of the present invention, such as stem cells, mesenchymal stem cells, Muse cells, ES cells, iPS cells, hair papilla cells, hair follicle stem cells, sebaceous gland stem cells, neural stem cells, olfactory nerve sheathing cells, bone marrow mesenchymal cells, dental pulp cells, Schwann cells, neural progenitor cells, dopaminergic neural progenitor cells, corneal stromal cells, corneal endothelial cells, corneal epithelial cells, retinal pigment epithelial cells, myoblasts, cardiac myocytes, nephron progenitor cells, differentiation cells, and the like. The cells are kidney cells, kidney stem cells, hepatocytes, hepatic progenitor cells, colon epithelial stem cells, colon absorptive epithelial cells, colon secretory epithelial cells, DFAT cells, vascular endothelial cells, vascular pericytes, smooth muscle cells, bone marrow mononuclear cells, chondroblasts, cultured cartilage, epidermal cells, epidermal stem cells, skin fibroblasts, dental pulp (stem) cells, ameloblasts, periodontal ligament stem cells, osteoblasts, osteoclasts, dendritic cells (activated), mononuclear cells, T cells, CAR-T cells, γδT cells, NK cells, CAR-NK cells, hematopoietic stem cells, platelets, plasma, or red blood cells. The cells may be in the form of a cell sheet or a sheet-like cell culture that has been cultured to form a sheet-like tissue. EXAMPLES

[0132] The present invention will be described in detail with reference to the following examples, but the present invention is not limited thereto. In the following, the hyaluronic acid manufactured by Shanghai Easier Industrial Development Co., Ltd. is sodium hyaluronate, but for the sake of simplicity, it is referred to as "hyaluronic acid".

[0133] <Preparation of cryopreservation agent> Example 1 In a pressure-resistant container with a volume of 2L, a mixture of high molecular weight hyaluronic acid (manufactured by Shanghai Easier Industrial Development Co., Ltd.) with an average molecular weight of 1 million and water is mixed at a ratio of 20:100, and the mixture is subjected to subcritical treatment at a treatment temperature of 175°C, a treatment pressure of 0.89MPa, and a treatment time of 3 minutes.Then, the subcritical treatment product is dried by freeze-drying or spray-drying.As a result, a mixture of high molecular weight hyaluronic acid with an intrinsic viscosity of 0.49dL / g and a viscosity average molecular weight of 10000 and low molecular weight hyaluronic acid with an intrinsic viscosity of 0.08dL / g and a viscosity average molecular weight of 1000, that is, the cryopreservation agent used in the present invention, is obtained.

[0134] <Preparation of cell preparations and evaluation of cryopreservation effects> Example 2 In 1 L of water for injection, 140 mg of calcium chloride, 100 mg of magnesium chloride hexahydrate, 100 mg of magnesium sulfate heptahydrate, 400 mg of potassium chloride, 60 mg of potassium dihydrogen phosphate, 350 mg of sodium bicarbonate, 48 mg of disodium hydrogen phosphate, 11 g of D(+)-glucose, 9 g of sodium chloride, 10 g of proline, and 100 g of the cryopreservation agent (solid content) of the present invention obtained in Example 1 were dissolved. Human retinal pigment epithelial (RPE) cells (H-RPE-Human Retinal Pigment Epithelial Cells, Lonza, Catalog No. 00194987) were added to the solution at a cell density of 1 × 10 6 The cells were suspended at a concentration of 10 cells / mL to obtain a cell preparation.

[0135] The cell preparations were frozen in a slow cell freezer (Nalgene® Mr. Frosty) in a −80° C. freezer. The frozen cell preparations were stored at −80° C. for 24 hours and then rapidly thawed in a 37° C. water bath. The cell viability of the cells immediately after thawing was assessed by trypan blue exclusion. The results are shown in FIG. 1.

[0136] The thawed cells were cultured in RtEGM™ BulletKit™ (Lonza) in a CO2 incubator controlled at 37°C and 5% CO2 for 5 days. The morphology of the cells after culture was observed under a microscope. As a control, RPE cells before cryopreservation were cultured in the same manner and observed under a microscope. The respective images are shown in Figure 2.

[0137] Comparative Example 1 As in Example 2, human retinal pigment epithelium (RPE) cells were cryopreserved at 1 × 10 in a DMSO-containing STEM-CELLBANKER (registered trademark) GMP grade (Zenoac Resources Co., Ltd.) (a mixture of 100 mL of DMSO, an aqueous solution in which 5 g of sodium carboxymethylcellulose (molecular weight 760,000) was dissolved in 750 mL of distilled water, and an aqueous solution in which 30.0 g of glucose, 0.8 g of sodium bicarbonate, 0.36 g of 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid, and 1.576 g of phosphate buffer were dissolved in 150 mL of distilled water). 6 The cells were suspended at a concentration of 100 / mL. The suspension was cryopreserved and the cell viability after thawing was examined in the same manner as in Example 2. The results are shown in Figure 1. Furthermore, the thawed cells were cultured and observed under a microscope in the same manner as in Example 2. The observed image is shown in Figure 2.

[0138] Comparative Example 2 A suspension was prepared in the same manner as in Comparative Example 1, except that propylene glycol-containing STEM-CELLBANKER (registered trademark) DMSO Free GMP grade (manufactured by Zenoac Resources Co., Ltd.) (a mixture of 100 mL of propylene glycol, an aqueous solution in which 5 g of sodium carboxymethylcellulose (molecular weight 760,000) was dissolved in 750 mL of distilled water, and an aqueous solution in which 30.0 g of glucose, 0.8 g of sodium bicarbonate, 0.36 g of 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid, and 1.576 g of phosphate buffer were dissolved in 150 mL of distilled water) was used as the cryopreservation solution, and the cell viability after cryopreservation and thawing was examined. The results are shown in FIG. 1. In addition, the thawed cells were cultured and observed under a microscope in the same manner as in Example 2. The observed image is shown in FIG. 2.

[0139] As shown in FIG. 1, the cell preparation of Example 2 containing RPE cells showed a cell viability after freezing and thawing that was almost equivalent to that of Comparative Example 1, which used DMSO as a cryoprotectant. Moreover, the cell viability of Example 2 was significantly higher than that of Comparative Example 2, which used a cryoprotectant that did not contain DMSO. It can be seen that the cell preparation of the present invention is less susceptible to a decrease in viability due to cryopreservation. Since a protective effect almost equivalent to that of Comparative Example 1, which uses DMSO, was obtained, it can be seen that the cell preparation of the present invention can be well cryopreserved. Although not shown in FIG. 1, the cell survival rate in Example 4 described below is expected to be approximately the same as that in Comparative Example 1.

[0140] As shown in Figure 2, no significant differences were observed in the proliferation and morphology of RPE cells after thawing between Example 2, Example 4, Comparative Example 1 (containing DMSO), and Comparative Example 2 (containing propylene glycol). In addition, by comparing with the morphology of RPE cells before freezing (unfrozen), it was confirmed that there was no change in cell morphology before and after freezing in Example 2 and Example 4.

[0141] Example 3 A cell preparation was prepared in the same manner as in Example 2 using third passage normal canine mesenchymal stem cells derived from canine bone marrow (cyagen, catalog number: CAXMX-01001).

[0142] The cell preparations were frozen in a slow cell freezer (Nalgene® Mr. Frosty) at -80°C and then stored in the -80°C freezer for the specified periods shown below. The day after freezing (24 hours later), the cells were rapidly thawed in a 37°C warm bath. The cell viability of the cells immediately after thawing was evaluated by trypan blue exclusion. The results are shown in Figure 3.

[0143] The cells were thawed after 20 days of cryopreservation and cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS / FCS), 100 unit / mL penicillin, and 100 μg / mL streptomycin for 7 days in a CO2 incubator controlled at 37°C and 5% CO2. The morphology of the cells after culture was observed under a microscope. The observed images are shown in Figure 4.

[0144] The cells that had reached confluence through culture were used to differentiate into bone cells by culturing them in osteogenic differentiation induction medium (MSCgo™ Rapid Osteogenic XF (Biological Industries, Beit Haemek, Israel) for approximately 3 weeks in a CO2 incubator controlled at 37°C and 5% CO2. Calcification (calcium deposition) was stained using the Alizarin Red S staining method as an indicator of bone cells. The results are shown in Figure 5A.

[0145] In addition, the cells cultured to confluence were differentiated into adipocytes by culturing for about 3 weeks in MSCgo™ Adipogenic XF (Biological Industries, Beit Haemek, Israel) containing MSCgo™ Adipogenic- SF,XF Supplement Mix-1 and MSCgo™ Adipogenic- SF,XF Supplement Mix-2. Differentiation was confirmed by staining lipid droplets in adipocytes with Oil Red O staining. The results are shown in Figure 5B.

[0146] In addition, when the cells reached 80-90% confluence through culture, 1 × 10 5The cells were seeded at 100 cells / well and cultured for 24 hours in Dulbecco's modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS / FCS), 100 unit / mL penicillin, and 100 μg / mL streptomycin to form a cell pellet. The cell pellet was then cultured for approximately 3 weeks in MSCgo™ Chondrogenic XF, Human (Biological Industries, Beit Haemek, Israel) containing MSCgo™ Chondrogenic XF Supplement Mix to differentiate into chondrocytes. Mucopolysaccharides were stained with Alcian blue to confirm differentiation into chondrocytes. The results are shown in Figure 5C.

[0147] In addition, cells were thawed after 20 days of cryopreservation and plated in 12-well plates at 2 × 10 5 The next day, the medium was replaced with Dulbecco's modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS / FCS), 100 unit / mL penicillin, and 100 μg / mL streptomycin. Three days later, the concentrations of HGF, IL-10, and VEGF in the cell lysates were quantified. The quantification of HGF, IL-10, and VEGF was performed using dedicated kits (catalog number SEA056Ca, Cloud-Clone (IL-10 measurement), catalog number SEA047Ca, Cloud-Clone (HGF measurement), catalog number CAVE00, R&D Systems (VEGF measurement)) according to the procedure in the kit. The measured values ​​were normalized to the total protein concentration in the cell lysates measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) as an internal standard. The quantitative results of HGF and IL-10 are shown in Figures 6A and 6B, respectively.

[0148] Example 4 (1) In a 2L pressure vessel, polymeric hyaluronic acid (manufactured by Shanghai Easier Industrial Development Co., Ltd.) with an average molecular weight of 1 million was mixed with water in a ratio of 20:100, and subcritical processing was carried out at a processing temperature of 175°C, a processing pressure of 0.89MPa, and a processing time of 7 minutes. The subcritically processed product was then dried by freeze drying or spray drying. This resulted in hyaluronic acid with an intrinsic viscosity of 0.08dL / g and a viscosity average molecular weight of approximately 1000. (2) In 1 L of water for injection, 140 mg of calcium chloride, 100 mg of magnesium chloride hexahydrate, 100 mg of magnesium sulfate heptahydrate, 400 mg of potassium chloride, 60 mg of potassium dihydrogen phosphate, 350 mg of sodium bicarbonate, 48 mg of disodium hydrogen phosphate, 11 g of D(+)-glucose, 9 g of sodium chloride, 10 g of proline, 90 g of pullulan (manufactured by Tokyo Chemical Industry Co., Ltd.) with a viscosity average molecular weight of 373,000, and 10 g of hyaluronic acid with a viscosity average molecular weight of 1,000 obtained in (1) were dissolved. The total amount of the cryopreservative pullulan and hyaluronic acid was 100 g. (3) Human retinal pigment epithelial (RPE) cells (H-RPE-Human Retinal Pigment Epithelial Cells, Lonza, Catalog No.: 00194987) were added to the solution obtained in (2) at a cell density of 1 × 10 6 The cells were suspended at a concentration of 10 cells / mL to obtain a cell preparation. (4) The obtained cell preparation was frozen in a slow cell freezer (Nalgene® Mr. Frosty) in a −80° C. freezer. The frozen cell preparation was stored at −80° C. for 24 hours and then rapidly thawed in a 37° C. warm bath. The cell viability of the cells immediately after thawing was evaluated by trypan blue exclusion. The viability was 90%. The thawed cells were cultured and observed under a microscope in the same manner as in Example 2. The observed images are shown in FIG.

[0149] Comparative Example 3 Hanks' balanced salt solution (HBSS) (Gibco) containing 10% DMSO was used as the cryopreservation solution. The main components of HBSS are as follows: calcium chloride 140 mg, magnesium chloride hexahydrate 100 mg, magnesium sulfate heptahydrate 100 mg, potassium chloride 400 mg, potassium dihydrogen phosphate 60 mg, sodium bicarbonate 350 mg, disodium hydrogen phosphate 48 mg, D(+)-glucose 1 g, and sodium chloride 8 g per 1 L of aqueous solution.

[0150] Normal canine mesenchymal stem cells were suspended in this cryopreservation solution, and the suspension was cryopreserved, and the cell survival rate after thawing was examined the day after freezing (24 hours later), as in Example 2. The results are shown in Figure 3. Furthermore, as in Example 3, the thawed cells were cultured and observed under a microscope. An image of the cultured cells is shown in Figure 4.

[0151] Moreover, differentiation into osteocytes, adipocytes, and chondrocytes was performed in the same manner as in Example 3. The results are shown in Figures 5A to 5C, respectively. Moreover, the concentrations of HGF, IL-10, and VEGF were quantified in the same manner as in Example 3. The quantitative results of HGF and IL-10 are shown in Figures 6A and 6B, respectively.

[0152] Comparative Example 4 A suspension was prepared in the same manner as in Comparative Example 3, except that DMSO-containing STEM-CELLBANKER (registered trademark) GMP grade (manufactured by Zenoac Resources Co., Ltd.) was used as the cryopreservation solution, and the cell viability after cryopreservation and thawing was examined. The results are shown in Figure 3. Furthermore, the thawed cells were cultured and observed under a microscope in the same manner as in Example 3. An image of the cultured cells is shown in Figure 4.

[0153] Moreover, differentiation into osteocytes, adipocytes, and chondrocytes was performed in the same manner as in Example 3. The results are shown in Figures 5A to 5C, respectively. Moreover, the concentrations of HGF, IL-10, and VEGF were quantified in the same manner as in Example 3. The quantitative results of HGF and IL-10 are shown in Figures 6A and 6B, respectively.

[0154] Comparative Example 5 A suspension was prepared in the same manner as in Comparative Example 3, except that propylene glycol-containing STEM-CELLBANKER (registered trademark) DMSO Free GMP grade (manufactured by Zenoac Resources Co., Ltd.) was used as the cryopreservation solution, and the cell survival rate after cryopreservation and thawing was examined. The results are shown in Figure 3. Furthermore, the thawed cells were cultured and observed under a microscope in the same manner as in Example 3. An image of the cultured cells is shown in Figure 4.

[0155] Moreover, differentiation into osteocytes, adipocytes, and chondrocytes was carried out in the same manner as in Example 3. The results are shown in Figures 5A to 5C, respectively.

[0156] Comparative Example 6 Canine bone marrow-derived normal canine mesenchymal stem cells, which had been cryopreserved in liquid nitrogen using 10% DMSO in the second passage, were passaged (third passage) and used as unfrozen cells. Observation images of the cultured cells are shown in FIG. 4. Differentiation into osteocytes, adipocytes, and chondrocytes was performed in the same manner as in Example 3. The results are shown in FIGS. 5A to 5C, respectively. Quantification of HGF, IL-10, and VEGF concentrations was also performed in the same manner as in Example 3. Quantitative results of HGF and IL-10 are shown in FIGS. 6A and 6B, respectively.

[0157] As shown in FIG. 3, the normal canine mesenchymal stem cells of the cell preparation of Example 3 showed a cell viability after freezing and thawing that was almost equivalent to that of Comparative Examples 3 and 4, which used DMSO as a cryoprotectant. This cell viability was significantly higher than that of the cryoprotectant of Comparative Example 5, which did not contain DMSO. It can be seen that the cell preparation of the present invention is less susceptible to a decrease in viability due to cryopreservation. Since a protective effect almost equivalent to that of Comparative Examples 3 and 4, which used DMSO, was obtained, it can be seen that the cell preparation of the present invention can be well cryopreserved.

[0158] As shown in Figure 4, the normal canine mesenchymal stem cells after thawing in Example 3 maintained a morphology similar to that in Comparative Examples 3 to 6, indicating that the fibroblast-like cell morphology specific to normal canine mesenchymal stem cells was equally maintained.

[0159] As shown in Figures 5A to 5C, it can be seen that the normal canine mesenchymal stem cells after thawing in Example 3 and the unfrozen cells (Comparative Example 6) were well differentiated into bone cells, adipocytes, and chondrocytes. On the other hand, in Comparative Examples 3 and 4, which were cryopreserved with the addition of DMSO, the differentiation of the thawed cells into bone cells was hardly observed. This result indicates that the addition of DMSO, an intracellularly penetrating cryoprotectant, to the cell preparation affects the differentiation ability of the cells in the cell preparation after thawing. It can be seen that the differentiation ability of the cells is well maintained in the cell preparation of the present invention even after cryopreservation.

[0160] As shown in Figure 6A, a comparison of the normal canine mesenchymal stem cells after thawing in Example 3 with Comparative Examples 3, 4, and 6 shows that the amount of HGF in the cells in Example 3 is equal to or greater than that in Comparative Examples 3, 4, and 6.

[0161] As shown in Figure 6B, a comparison of the normal canine mesenchymal stem cells after thawing in Example 3 with Comparative Examples 3, 4, and 6 shows that the amount of IL-10 in the cells in Example 3 is equal to or higher than that in Comparative Examples 3, 4, and 6.

[0162] Intracellular VEGF was not detected in the thawed normal canine mesenchymal stem cells of Example 3 and Comparative Examples 3, 4, and 6. It can be seen that in Example 3 and Comparative Examples 3, 4, and 6, there is no change in VEGF production due to freezing, and it is equivalent after storage to that before storage. It can be seen that the amount of intracellular VEGF is unlikely to change due to cryopreservation in the cell preparation of the present invention.

[0163] <Analysis of CD antigen expression in canine mesenchymal stem cells after cryopreservation and thawing in cell preparations containing normal canine mesenchymal stem cells derived from canine adipose tissue> The cell preparation prepared in Example 3 containing the third passage canine adipose tissue-derived normal canine mesenchymal stem cells (collected from the adipose tissue of a healthy young dog) and Comparative Example 4 in which normal canine mesenchymal stem cells were suspended in DMSO-containing STEM-CELLBANKER (registered trademark) GMP grade (manufactured by Zenoac Resources Co., Ltd.) were frozen in a slow cell freezer (Nalgene (registered trademark) Mr. Frosty) in a -80°C freezer, and then frozen and stored in the -80°C freezer for 4 days. After 4 days, the frozen cell preparation and suspension were removed and rapidly thawed in a 7°C warm bath. In the thawed cell suspension, the canine adipose tissue-derived mesenchymal stem cells were stained with fluorescently labeled antibodies corresponding to cell surface antigens (positive markers: CD44, CD90, and CD29, negative markers: CD45, and MHC class II) and analyzed by flow cytometry. The results are shown in Figure 7.

[0164] The analysis was carried out as follows.

[0165] First, 5×10 5The suspension containing 100 cells was added to an Eppendorf tube, and 1 mL of PBS(-) (Nacalai Tesque, Kyoto, Japan) (FACS buffer) containing 1% albumin (Fujifilm-Wako, Osaka, Japan) was added. The tube was centrifuged at 300g for 5 minutes, the supernatant was removed, and 100 μL of each antibody solution was added and incubated for 30 minutes on ice in the dark. The labeled antibodies used were CD44-FITC (catalog number: 11-5440-42), CD29-FITC (catalog number: 11-0299-42), and CD90-PE (catalog number: 12-5900-42) as positive markers expressed in mesenchymal stem cells, and CD45-FITC (catalog number: 11-5450-42) and MHC class II-FITC (catalog number: 11-5909-41) as negative markers not expressed in mesenchymal stem cells. In addition, the recommended isotype control was used as a fluorescence control. All antibodies were purchased from Invitrogen (US) and used at the recommended concentration. After antibody reaction, 900 μL of FACS buffer was added to remove excess unbound antibodies, and the mixture was centrifuged at 300 g for 4 minutes to remove the supernatant. 500 μL of FACS buffer was added to this and resuspended to prepare the analysis sample. The analysis sample was added to a test tube (352052; Corning, NY) through a mesh to remove debris. Analysis was performed using a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA) to acquire data for a total of 10,000 events for each sample. Data analysis was performed using FACSDiva software (BD Biosciences, San Jose, CA).

[0166] In addition, the cell surface antigens of the unfrozen cells of Comparative Example 6, which were obtained by passage culture of normal canine mesenchymal stem cells derived from canine adipose tissue (third passage), were analyzed in the same manner using the same labeled antibody. The results are shown in FIG.

[0167] Comparative Example 7 A suspension of normal canine mesenchymal stem cells was prepared in the same manner as in Comparative Example 4, except that CELLBANKER (registered trademark) 1 plus (manufactured by Zenoac Resources Co., Ltd.), a serum-type cell preservation solution containing DMSO, was used as the freezing preservation solution. The prepared suspension was frozen in a slow cell freezer (Nalgene (registered trademark) Mr. Frosty) in a -80°C freezer, and frozen and stored in the -80°C freezer for 4 days. The frozen suspension was then removed and rapidly thawed in a 37°C warm bath. After thawing, cell surface antigens were analyzed in the same manner using the same labeled antibody. The results are shown in Figure 7.

[0168] As shown in Figure 7, the cells of Example 3 were positive for all of the positive markers CD44, CD29, and CD90, and were negative for all of the negative markers CD45 and MHC class II. Therefore, it is understood that in the cell preparation of the present invention, the mesenchymal stem cells derived from canine adipose tissue are preserved as mesenchymal stem cells and maintained in an undifferentiated state.

[0169] From the above results, it can be seen that the cell preparation of the present invention significantly suppresses the decline in cell function and regenerative ability associated with cryopreservation. The cell preparation of the present invention can provide cells that need to be maintained in an undifferentiated state, such as stem cells for regenerative medicine, without the risk of differentiation. It is believed that the cell preparation of the present invention can provide cells that exhibit homogeneous and stable performance.

Claims

1. A polymer having a viscosity average molecular weight of more than 3,000 and not more than 500,000, the polymer including a monomer having a hydrophilic group as a repeating unit, or a salt thereof; A saccharide or a salt thereof having a viscosity average molecular weight of 3,000 or less; The cells, A pharma- ceutically acceptable carrier and 1. A cell preparation comprising:

2. The cell preparation according to claim 1, wherein the pharma- ceutical acceptable carrier is at least one selected from the group consisting of a buffering agent, a stabilizer and an isotonicity agent.

3. The cell preparation according to claim 2 , wherein the pharma- ceutically acceptable carrier comprises an isotonicity agent and a buffering agent.

4. The cell preparation according to any one of claims 1 to 3, further comprising an amino acid.

5. The cell preparation according to any one of claims 1 to 4, further comprising a monosaccharide.

6. The cell preparation according to any one of claims 1 to 5, wherein the hydrophilic group of the monomer having a hydrophilic group is at least one selected from the group consisting of a hydroxyl group, a carboxylic acid group, and a salt thereof.

7. The cell preparation according to any one of claims 1 to 6, wherein the polymer further comprises, as a repeating unit, a nitrogen-containing monomer having an optionally substituted amino group or an optionally substituted amide group.

8. The cell preparation according to claim 7, wherein the polymer is an alternating copolymer of the monomer having the hydrophilic group and the nitrogen-containing monomer.

9. The cell preparation according to any one of claims 1 to 8, wherein the monomer having a hydrophilic group has a hydroxyl group substituted at an equatorial position.

10. The cell preparation according to any one of claims 1 to 9, wherein the polymer has a viscosity average molecular weight of 5,000 or more.

11. The cell preparation according to any one of claims 1 to 10, wherein the polymer comprises a plurality of sugar residues.

12. The cell preparation according to any one of claims 1 to 11, wherein the saccharide is a monosaccharide, a disaccharide, or an oligosaccharide.

13. The cell preparation according to claim 12, wherein the sugar is glucose, fructose, galactose, or uronic acid in which the alcohol group of these sugars is oxidized, or an amino sugar in which the alcohol group is substituted with an amino group, sucrose, a cleavage product of a glycosaminoglycan, a constituent monosaccharide of a glycosaminoglycan, or a polymer or combination thereof.

14. The cell preparation according to claim 13, wherein the saccharide is glucuronic acid or N-acetylglucosamine.

15. The cell preparation according to any one of claims 2 to 14, wherein the isotonicity agent comprises an inorganic salt.

16. The cell preparation according to claim 15, wherein the inorganic salt is a chloride.

17. The cell preparation according to any one of claims 2 to 16, wherein the buffer is a phosphate or a bicarbonate.

18. The cell preparation according to any one of claims 4 to 17, wherein the amino acid is proline.

19. The cell preparation according to any one of claims 5 to 18, wherein the monosaccharide is glucose.

20. The cell preparation according to any one of claims 1 to 19, further comprising a protein activator.

21. The cell preparation of claim 20, wherein the protein activator is a magnesium salt or a calcium salt.

22. The cell preparation according to any one of claims 1 to 21, wherein the concentration of the polymer in the cell preparation is 5 w / v % or more and 20 w / v % or less.

23. The cell preparation according to any one of claims 1 to 22, wherein the concentration of the sugar in the cell preparation is 0.1 w / v % or more and 10 w / v % or less.

24. The cell preparation according to any one of claims 1 to 23, which is in the form of a cell sheet or a sheet-like cell culture.

25. The cell preparation according to any one of claims 1 to 24, which is in a frozen state.

26. The cell preparation of claim 25, wherein the cell preparation in a frozen state comprises a plurality of cryopreserved cells, the cells having at least 80% viability at the time of being frozen and recovered following thawing.

27. The cell preparation according to any one of claims 1 to 26, wherein the cells are stem cells or cells derived from stem cells.

28. The cells include stem cells, mesenchymal stem cells, Muse cells, ES cells, iPS cells, hair papilla cells, hair follicle stem cells, sebaceous gland stem cells, neural stem cells, olfactory nerve sheathing cells, bone marrow mesenchymal cells, dental pulp cells, Schwann cells, neural progenitor cells, dopaminergic neural progenitor cells, corneal stromal cells, corneal endothelial cells, corneal epithelial cells, retinal pigment epithelial cells, myoblasts, cardiac myocytes, nephron progenitor cells, differentiated kidney cells, kidney stem cells, hepatic cells, hepatic progenitor cells, colon epithelial stem cells, colon absorptive system epithelial cells, colon secretory cells, and the like. The cell preparation according to any one of claims 1 to 27, which is an endothelial cell, a DFAT cell, a vascular endothelial cell, a vascular pericyte, a smooth muscle cell, a bone marrow mononuclear cell, a chondroblast, a cultured cartilage, an epidermal cell, an epidermal stem cell, a skin fibroblast, a dental pulp (stem) cell, an ameloblast, a periodontal ligament stem cell, an osteoblast, an osteoclast, a dendritic cell (activated), a mononuclear cell, a T cell, a CAR-T cell, a γδT cell, a NK cell, a CAR-NK cell, a hematopoietic stem cell, a platelet, a plasma, or a red blood cell.

29. The cell preparation according to claim 24, wherein the cell sheet is a corneal epithelial sheet, a retinal pigment sheet, a myocardial cell sheet, a hepatic cell sheet, an epidermis sheet, a cartilage sheet, a periosteum sheet or a stem cell sheet.

30. The cell preparation according to any one of claims 1 to 29, which is for the treatment and / or prevention of nerve disorder, skin defect, cerebrovascular disorder, spinal cord injury, brain trauma, central nervous system disease, Parkinson's disease, corneal disorder, Stevens-Johnson syndrome, ocular pemphigoid, corneal chemical injury / burn, cicatricial keratoconjunctival epitheliopathy, bullous keratopathy, infectious keratitis, corneal dystrophy, retinal optic neuropathy, corneal epithelial stem cell deficiency, liver disorder, hyperammonemia, ischemic heart disease, heart failure, thrombocytopenia, arteriosclerosis obliterans, burns, wounds, cartilage damage, complications during bone marrow transplantation, aplastic anemia, metabolic liver disease, colitis, arteriosclerosis obliterans, ischemia, peripheral arterial disease, non-arteriosclerosis, bone system disease, osteoarthritis, bone tumor, bone necrosis, lower limb ischemic disease, cancer, rheumatism, osteoporosis, or muscular dystrophy.

31. The cell preparation according to any one of claims 1 to 30, which is administered intrabone marrow, intravenously, intramuscularly, intraperitoneally, intraocularly, or intracerebrally.

32. A polymer having a viscosity average molecular weight of more than 3,000 and not more than 500,000, the polymer including a monomer having a hydrophilic group as a repeating unit, or a salt thereof; A saccharide or a salt thereof having a viscosity average molecular weight of 3,000 or less; A pharma- ceutically acceptable carrier and 23. A solution for storing cells comprising:

33. 33. The solution of claim 32, further comprising an amino acid.

34. 34. The solution according to claim 32 or 33, further comprising a monosaccharide.

35. A polymer having a viscosity average molecular weight of more than 3,000 and not more than 500,000, the polymer including a monomer having a hydrophilic group as a repeating unit, or a salt thereof; A saccharide or a salt thereof having a viscosity average molecular weight of 3,000 or less; A pharma- ceutically acceptable carrier and 23. Use of a solution comprising:

36. The cells are selected from the group consisting of stem cells, mesenchymal stem cells, Muse cells, ES cells, iPS cells, hair papilla cells, hair follicle stem cells, sebaceous gland stem cells, neural stem cells, olfactory nerve sheathing cells, bone marrow mesenchymal cells, dental pulp cells, Schwann cells, neural progenitor cells, dopaminergic neural progenitor cells, corneal stromal cells, corneal endothelial cells, corneal epithelial cells, retinal pigment epithelial cells, myoblasts, cardiac myocytes, nephron progenitor cells, differentiated kidney cells, kidney stem cells, hepatic cells, hepatic progenitor cells, colon epithelial stem cells, colon absorptive system epithelial cells, and the like. The use according to claim 35, wherein the target cell is a human fibroblast, a human colon secretory epithelial cell, a DFAT cell, a vascular endothelial cell, a vascular pericyte, a smooth muscle cell, a bone marrow mononuclear cell, a chondroblast, a cultured cartilage, an epidermal cell, an epidermal stem cell, a skin fibroblast, a dental pulp (stem) cell, an ameloblast, a periodontal ligament stem cell, an osteoblast, an osteoclast, a dendritic cell (activated), a mononuclear cell, a T cell, a CAR-T cell, a γδT cell, a NK cell, a CAR-NK cell, a hematopoietic stem cell, a platelet, a plasma, or a red blood cell.

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