Cryopreservation liquid

Abstract

To provide a cryopreservation liquid for hematopoietic cells with a high cell survival rate, and a cryopreservation method for hematopoietic cells.SOLUTION: A cryopreservation liquid for hematopoietic cells includes, in a solvent: a polymer having a viscosity average molecular weight of more than 3000 and 500000 or less, the polymer including a monomer having a hydrophilic group as a repeat unit, or a salt thereof; a saccharide having a viscosity average molecular weight of 3000 or less, or a salt thereof; and a polyhydric alcohol. There is also provided a cryopreservation method for hematopoietic cells using the cryopreservation liquid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a cryopreservation solution, particularly a cryopreservation solution for blood cells, and a method for cryopreserving blood cells using the cryopreservation solution. [Background technology]

[0002] With the dramatic advances in regenerative medicine research in recent years, cell therapy and other regenerative medicine approaches are being actively pursued not only in humans but also in veterinary medicine. Bone marrow-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells are harvested from living organisms and then expanded in large quantities for use in the aforementioned regenerative medicine and regenerative medicine research. In this case, it is common for excess cells to be cryopreserved and used as needed. There is also a growing demand for a stable supply of such cells.

[0003] In the cryopreservation mechanism of cells, it is known that when ice crystals grow inside cells during the freezing and / or thawing process, the cell membrane and intracellular structures are damaged, and cellular proteins are denatured, resulting in fatal damage to the cells.

[0004] To prevent such intracellular freezing, methods of cryopreserving cells have been used, such as vitrification, which uses a high concentration of cryoprotectant to prevent the formation of ice crystals, and slow freezing, which involves slowly cooling a physiological solution containing cells and a cryoprotectant.

[0005] Dimethyl sulfoxide, for example, is a widely used cryoprotectant. Examples of cryopreservation solutions optimized for the cryopreservation of stem cells include STEM-CELLBANKER (registered trademark) (Zenoac Resources, Inc.) for slow freezing.

[0006] Patent Document 1 describes a cryopreservation solution for biological samples, which contains, in a solvent, a polymer or a salt thereof, the polymer having a viscosity-average molecular weight of more than 3,000 and not more than 500,000, the polymer containing a monomer having a hydrophilic group as a repeating unit, and a saccharide or a salt thereof having a viscosity-average molecular weight of not more than 3,000. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2020 / 166711 Summary of the Invention [Problem to be solved by the invention]

[0008] Patent Document 1 describes that in cryopreservation using the cryopreservation solution of Patent Document 1, biological samples can be cryopreserved while maintaining the properties of the cells, without using substances such as dimethyl sulfoxide, which have been widely used as cryoprotectants but are known to have cytotoxicity and differentiation-inducing properties. However, depending on the type of biological sample, there are cases where a sufficient cryopreservation effect cannot be obtained.

[0009] In particular, when blood cells are used as biological samples for cryopreservation, the survival rate of the cells after thawing may be lower than that of mesenchymal stem cells, etc. Therefore, there is a need for the development of a cryopreservation solution that can provide a high cryopreservation effect even for such blood cells.

[0010] The present invention has been made in consideration of the above-mentioned problems and provides a cryopreservation solution for appropriately cryopreserving blood cells. In particular, it is an object of the present invention to provide a highly biocompatible cryopreservation solution for blood cells that can stably cryopreserve blood cells while maintaining high cell viability, without the addition of cytotoxic or differentiation-inducing cryoprotectants such as dimethyl sulfoxide. [Means for solving the problem]

[0011] The present invention relates to a cryopreservation solution for blood cells, comprising a polymer or its salt having a viscosity-average molecular weight of more than 3,000 and not more than 500,000, the polymer containing a repeating unit of a monomer having a hydrophilic group, a saccharide or its salt having a viscosity-average molecular weight of not more than 3,000, and a polyhydric alcohol in a solvent. The salt of the polymer or saccharide is preferably a metal salt, a halogen salt, or a sulfate. The metal salt is preferably a salt of an alkali metal or alkaline earth metal. Examples of alkali metals or alkaline earth metals include sodium, potassium, and calcium. Examples of halogens that can be used include chlorine and bromine.

[0012] The polyhydric alcohol preferably has at least three hydroxyl groups.

[0013] In the present invention, it is desirable that the polymer or its salt, which has a viscosity-average molecular weight of more than 3,000 and not more than 500,000 and contains a monomer having a hydrophilic group as a repeating unit, is contained as the main component, and the saccharide or its salt, which has a viscosity-average molecular weight of not more than 3,000, is contained as the secondary component. In this specification, the term "main component" refers to the component with the highest weight ratio among the components dissolved in the solvent. Components other than the main component are secondary components.

[0014] In the polymers used in the present invention, the hydrophilic groups contained in the repeating units of the monomers are preferably unmodified, or, if modified, the hydrophilic groups are preferably less than 50% of the total number of hydrophilic groups. The hydrophilic groups in the polymer are presumed to be involved in the vitrification of the solvent, the replacement of the water surrounding the biological sample with sugars of molecular weight 3000 or less, and the protection of the frozen cells. Therefore, if the hydrophilic groups are modified to become hydrophobic, the effectiveness of cryopreservation of cells decreases. Therefore, it is desirable to avoid hydrophobic polymers such as carboxypolyamino acids as the main component.

[0015] The cryopreservation solution for hematopoietic cells of the present invention preferably does not contain dimethyl sulfoxide, a cryoprotectant that can function as a differentiation factor. Furthermore, the cryopreservation solution for hematopoietic cells of the present invention preferably does not contain cytotoxic cryoprotectants such as ethylene glycol, as these 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 polymer or its salt is easy to handle as a cryopreservation solution.

[0017] A preferred cryopreservation solution for blood cells is one in which the monomer having a hydrophilic group is a monomer having a hydrophilic group that is at least one selected from the group consisting of a hydroxyl group and a carboxylic acid group and salts thereof.

[0018] A preferred cryopreservation solution for blood cells 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.

[0019] A preferred cryopreservation solution for blood cells is one in which the polymer is an alternating copolymer of the monomer having a hydrophilic group and the nitrogen-containing monomer.

[0020] A preferred cryopreservation solution for blood cells is one in which the monomer having a hydrophilic group is a monomer having a hydroxyl group substituted at an equatorial position.

[0021] A cryopreservation solution for blood cells containing a polymer having a viscosity average molecular weight of 5000 or more or a salt thereof is preferred.

[0022] A preferred cryopreservation solution for blood cells is one in which the polymer contains multiple sugar residues.

[0023] A cryopreservation medium for blood cells in which the saccharide is a monosaccharide, a disaccharide, or an oligosaccharide is preferred.

[0024] A cryopreservation solution for blood cells in which the sugar is glucose, fructose, galactose, or uronic acid in which the alcohol group of any of these is oxidized, or an amino sugar in which the alcohol group is substituted with an amino group, sucrose, a cleavage product of glycosaminoglycan, a constituent monosaccharide of glycosaminoglycan, or a polymer or combination thereof is preferred.

[0025] A cryopreservation medium for blood cells in which the sugar is glucose, glucuronic acid, or N-acetylglucosamine is preferred.

[0026] A preferred cryopreservation solution for blood cells is one in which the polyhydric alcohol has at least three hydroxyl groups.

[0027] A cryopreservation solution for blood cells in which the concentration of the polymer or its salt in the solution is 0.1 w / v % or more and 50 w / v % or less is preferred.

[0028] A cryopreservation solution for blood cells in which the concentration of sugars or salts thereof is 0.1 w / v % or more and 10 w / v % or less is preferred.

[0029] A cryopreservation solution for blood cells in which the concentration of polyhydric alcohol in the cryopreservation solution is 1 w / v % or more and 60 w / v % or less is preferred.

[0030] A cryopreservation medium for blood cells is preferred in which the blood cells are megakaryocyte precursor cells.

[0031] A cryopreservation medium for blood cells is preferred, where the blood cells are granulocytes, lymphocytes, monocytes, or erythrocytes.

[0032] A cryopreservation solution for blood cells is preferably one in which the blood cells are white blood cells.

[0033] The present invention also relates to a method for cryopreserving blood cells, which comprises suspending the blood cells in any of the above-mentioned cryopreservation solutions and cryopreserving them.

[0034] The "viscosity average molecular weight" of the polymer or saccharide used in the present invention can be determined by the following method and calculation formula.

[0035] Intrinsic viscosity measurement: (1) Dissolve a predetermined 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 a polymer or sugar sample in a standard solution at 30°C. Measure the viscosity of both the standard solution and the stock solution, and adjust the viscosity of the stock solution relative to the standard solution to 2.0 to 2.4. (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 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: TIFF2025120359000001.tif2146where 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 line. The intercept of the approximate line (where the polymer or sugar concentration is 0) is the limiting viscosity.

[0036] Viscosity average molecular weight: The viscosity average molecular weight is calculated from the intrinsic viscosity. TIFF2025120359000002.tif3559The viscosity average molecular weight M is calculated from the above Mark Hoying-Sakurada equation using the intrinsic viscosity derived from the measurement and the values of K and α published in literature, etc.

[0037] 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" (edited by the Society of Polymer Science, Incorporated Association). The viscosity average molecular weight can be calculated using these published values.

[0038] For example, in the case of hyaluronic acid, K = 3.6 × 10 -4 and α = 0.78. For pullulan and gelatin, K = 9 × 10 from the literature. -4 , α=0.5. For example, in the case of dextran, K=6.3×10 -8 , α = 1.4, K = 5.8 × 10 for chondroitin sulfate -4 , α=0.74 can be used.

[0039] 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 hypothetical viscosity average molecular weight.

[0040] The solvent used to prepare the cryopreservation solution of the present invention is preferably an aqueous solvent such as water. It is particularly preferable to use an isotonic solution in which the salt concentration and sugar concentration are adjusted with sodium ions, potassium ions, calcium ions, etc. to approximately match the osmotic pressure of body fluids or cellular fluids. Specific examples of such a solvent include water, saline, buffered saline solutions such as phosphate buffered saline (PBS), Dulbecco's phosphate buffered saline, Tris buffered saline (TBS), and HEPES-buffered saline, balanced salt solutions such as Hank's balanced salt solution, Ringer's solution, lactate Ringer's solution, acetate Ringer's solution, and bicarbonate Ringer's solution, as well as basal animal cell culture media such as D-MEM, E-MEM, αMEM, RPMI-1640 medium, Ham's F-12, Ham's F-10, and M-199, and other commercially available media.

[0041] The solvent may also contain calcium chloride, magnesium chloride, magnesium sulfate, potassium chloride, potassium dihydrogen phosphate, sodium hydrogen carbonate, disodium hydrogen phosphate, glucose, sodium chloride, an amino acid, etc. Proline is preferably selected as the amino acid. [Effects of the Invention]

[0042] The cryopreservation solution for hematopoietic cells of the present invention prevents damage to the cell membrane of hematopoietic cells during cryopreservation and achieves high survival and proliferation rates after thawing that are applicable to advanced cell therapy. This is due to the vitrification capabilities provided by the polymeric component with a viscosity-average molecular weight exceeding 3,000 and not exceeding 500,000, the inhibition of ice crystal formation and growth near the cell membrane and the protective effect of the low-molecular-weight sugars with a viscosity-average molecular weight of not more than 3,000, and the anti-freeze function of the polyhydric alcohol, which is intracellularly permeable and displaces intracellular water molecules. The cryopreservation solution for hematopoietic cells of the present invention exhibits high cryopreservation efficacy for many types of cells, including hematopoietic cells, nucleated cells, and non-nucleated cells, which was difficult to achieve with conventional techniques, without using highly cytotoxic substances.

[0043] In other words, the cryopreservation solution for blood cells of the present invention does not contain highly cytotoxic substances such as DMSO, thereby suppressing adverse effects on cells and maintaining cell viability and quality during and after cryopreservation. However, it is possible to add potentially cytotoxic chemicals such as DMSO and ethylene glycol at low concentrations that do not impair cellular function.

[0044] Furthermore, because the cryopreservation solution for blood cells of the present invention does not contain serum and / or serum-derived proteins, the frozen cells will not be contaminated with bacteria or viruses. However, it is possible to add proteins that are not contaminated with bacteria or viruses.

[0045] Furthermore, cryopreservation of cells using the cryopreservation solution for blood cells of the present invention does not require a high cooling rate to protect the cells during cooling, nor does it require low temperatures such as those required for liquid nitrogen, making the freezing process simple and cost-effective. [Brief explanation of the drawings]

[0046] [Figure 1] FIG. 1 shows the cell viability of THP-1 human monocytic cultured cells thawed after cryopreservation, as determined by flow cytometry. DETAILED DESCRIPTION OF THE INVENTION

[0047] The cryopreservation solution for blood cells of the present invention is a cryopreservation solution for blood cells containing, in a solvent, a polymer or a salt thereof having a viscosity-average molecular weight of more than 3,000 and not more than 500,000, a saccharide or a salt thereof having a viscosity-average molecular weight of not more than 3,000, and a polyhydric alcohol. The polymer of the present invention contains a monomer having a hydrophilic group as a repeating unit.

[0048] The viscosity average molecular weight of the polymer or salt thereof of 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 polymer or salt thereof is easy to handle as a cryopreservation solution.

[0049] The "viscosity average molecular weight" of the polymer or saccharide of the present invention means a value calculated by the following method and formula.

[0050] 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 predetermined 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 solution, remove the solvent from the solution to obtain the solid portion. For mixed samples of polymers and sugars, or mixed samples containing multiple polymers or multiple sugars, separate and fractionate each substance, then remove the solvent from each substance to obtain the polymer or sugar sample. If the polymer and / or sugar are unknown, identify them using HPLC, LC-MS, LC-IR, or other methods. If multiple unknown polymers and / or sugars are included, separate and fractionate each component, identify the substance for each polymer and / or sugar using HPLC, LC-MS, LC-IR, or other methods, and calculate the viscosity-average molecular weight as described below. Even if the polymer or sugar contains impurities that do not affect the viscosity (e.g., metal salts), the mixture can be considered a 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 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: TIFF2025120359000003.tif2146where 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 line. The intercept of the approximate line (where the polymer or sugar concentration is 0) is the limiting viscosity. Viscosity average molecular weight: The viscosity average molecular weight is calculated from the intrinsic viscosity. TIFF2025120359000004.tif3559The viscosity average molecular weight M can be calculated from the above Mark Hoeing-Sakurada equation using the intrinsic viscosity determined by measurement and the K and α values published in literature. In the case of hyaluronic acid, K = 3.6 x 10 -4 and α = 0.78 to calculate the viscosity average molecular weight M. For the pullulan and gelatin used in the examples, K = 9 × 10 -4 , α=0.5, and for dextran, K=6.3×10 -8 , α = 1.4, and for chondroitin sulfate, K = 5.8 × 10 -4 , α = 0.74, for carboxypolylysine, K = 2.78 × 10 -5 , α=0.87 is used.

[0051] 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" (edited by the Society of Polymer Science, Incorporated Association). The viscosity average molecular weight is calculated using these published values.

[0052] In the present invention, the molecular weight calculated by this method is referred to as the viscosity average molecular weight. In the case of monosaccharides, disaccharides, and compounds that are considered to be monomolecular molecules, such as sucrose and glucuronic acid, the molecular weight is clearly specified from the structural formula, and therefore the molecular weight specified from the structural formula is treated as the viscosity average molecular weight.

[0053] The polymers contained in the cryopreservation solution for hematopoietic cells of the present invention, which have a specific molecular weight and numerous hydrophilic groups, trap water molecules of the solvent within the matrix formed by the polymer chains during the cooling process of the cryopreserved sample. Because the polymer chains contain hydrophilic groups, the molecular motion of the water solvent is restricted during cooling, allowing the water to solidify and / or freeze in a vitrified state without crystallizing. Because the action of the polymer chains dehydrates and vitrifies the interior of cells, cryopreservation using the cryopreservation solution for hematopoietic cells of the present invention eliminates the need for increased solute (cryoprotectant) concentration or increased cooling rate, as required by conventional vitrification methods. The action of the polymer chains in the present invention suppresses intracellular ice crystal formation, thereby reducing the osmotic shock experienced by cells during freezing, a problem associated with conventional vitrification methods that utilize the osmotic pressure difference between the inside and outside of cells to dehydrate and vitrify the interior of the cells. Furthermore, since recrystallization does not occur during thawing of frozen cells, thawing damage to the cells is likely to be minimal.

[0054] The viscosity-average molecular weight of the polymer of the present invention is greater than 3,000 and less than 500,000. This level of viscosity-average molecular weight stabilizes the amorphous, vitreous state in the frozen state. Cells are less susceptible to damage caused by cooling and freezing, allowing for stable cryopreservation. Therefore, the survival rate of cells, including blood cells, after thawing a cryopreserved biological sample is high. When the viscosity-average molecular weight of the polymer is 3,000 or less, vitrification may be difficult to achieve. Furthermore, when the viscosity-average molecular weight of the polymer is greater than 500,000, the viscosity significantly increases, and problems such as reduced solubility and foaming of the prepared solution may occur, making it difficult to handle. The viscosity-average molecular weight is preferably 5,000 or more. Furthermore, polymers with a viscosity-average molecular weight of 400,000 or less, or even 200,000 or less, are preferred, with 150,000 or less being particularly preferred. This is because the viscosity can be adjusted low, making the cryopreservation solution easy to handle.

[0055] The polymer of the present invention is a polymer containing a repeating unit of a monomer having a hydrophilic group. Examples of the hydrophilic group include a hydroxyl group and a carboxylic acid group and its salt. The polymer of the present invention may also contain a repeating unit of a nitrogen-containing monomer having an optionally substituted amino group or an optionally substituted amide group. Furthermore, the polymer of the present invention preferably has a hydroxyl group at an equatorial position within its structure. This is believed to enable the water solvent to be more effectively trapped within the matrix formed by the polymer chains during freezing.

[0056] An example of a monomer having a hydrophilic group is a sugar residue. In this case, the polymer of the present invention may be a polymer containing repeating units of sugar residues linked by glycosidic bonds, or derivatives thereof. Examples of sugar residues include, but are not limited to, monosaccharides, or monosaccharides in which the hydroxyl and / or hydroxymethyl groups of monosaccharides have been substituted, such as monosaccharides in which the hydroxyl and / or hydroxymethyl groups have been 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.

[0057] Monosaccharides include triose, tetrose, pentose, hexose, and heptose. For example, pentoses include ribose, arabinose, xylose, lyxose, xylulose, ribulose, and deoxyribose. Hexoses include glucose, mannose, galactose, fructose, sorbose, tagatose, fucose, fuculose, and rhamnose.

[0058] For example, monosaccharides substituted with a carboxyl group include uronic acid. Examples of uronic acids include glucuronic acid, iduronic acid, mannuronic acid, and galacturonic acid. Examples of monosaccharides substituted with an amino group include amino sugars. Examples of amino sugars include glucosamine, galactosamine, mannosamine, and muramic acid. Examples of monosaccharides substituted with an N-acetylamino group include N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, and N-acetylmuramic acid. Examples of monosaccharides substituted with a sulfooxy group include galactose-3-sulfate. Examples of monosaccharides with 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.

[0059] For example, the polymer of the present invention may be a polymer containing the above-mentioned monosaccharides as repeating units. For example, the polymer of the present invention may be a polymer containing an optionally substituted pentose, hexose, or uronic acid, or a combination thereof, as repeating units. The polymer 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 of the present invention may be a glycosaminoglycan. Alternatively, the polymer may be a sulfated polysaccharide in which one or more hydroxyl groups are substituted with sulfoxy groups. Examples of the polymer of the present invention include, but are not limited to, hyaluronic acid, dextran, pullulan, and chondroitin sulfate.

[0060] The polymers used in the present invention may be naturally occurring, chemically synthesized, or commercially available. Naturally occurring polymeric compounds or commercially available polymeric compounds with larger molecular weights may be subjected to hydrolysis, enzyme treatment, subcritical treatment, or other treatments to obtain cleavage products, and the molecular weights may be adjusted to produce the polymers of the present invention. Furthermore, each monomer may be naturally occurring, may be a naturally occurring monomer that has been modified or substituted, or may be chemically synthesized. For example, preferably, the monomers contained in the polymers of the present invention are biological components. It is believed that cryopreservation solutions containing the polymers have low cytotoxicity.

[0061] The hydrophilic groups of the polymer of the present invention are desirably unmodified, or if modified, they account for 50% or less of the total number of hydrophilic groups, i.e., no substituents are introduced into the polymer chain, or if they are introduced, they account for 50% or less of the total number of hydrophilic groups. It is believed that the hydrophilic groups of the polymer, particularly OH, NH, and COOH groups, contribute to the protection of frozen cells, the vitrification of the solvent, and the replacement of sugar with water around the cells. Therefore, it is believed that not modifying these functional groups is advantageous for improving the survival rate of cells after thawing.

[0062] Furthermore, the hydrophilic groups of the polymers of the present invention are thought to hold low-molecular-weight sugars through hydrogen bonds, and it is presumed that the presence of such polymers holding low-molecular-weight sugars around biological samples such as cells can promote the replacement of water molecules with sugars near the cell membrane. Therefore, if the hydrophilic groups are modified, the hydrophilic groups' ability to hold low-molecular-weight sugars is reduced, and even if low-molecular-weight sugars coexist, there is a risk that they will not sufficiently contribute to improving cell viability. Therefore, modifying OH groups or NH groups with carboxylic acids or the like may not be desirable.

[0063] The cryopreservation solution of the present invention contains a saccharide or its salt having a viscosity-average molecular weight of 3000 or less. This saccharide replaces water molecules near the cell membrane, suppressing the formation and growth of ice crystals near the cell membrane, thereby significantly suppressing cell membrane damage. That is, the saccharide or its salt used in the present invention can function as a component for cell protection. The saccharide 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, and more preferably 1000 or less.

[0064] Such sugars include, for example, the monosaccharides described above as monomers constituting the polymers of the present invention. For example, sugars include 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, trehalose, or polymers or combinations thereof. Furthermore, sugars may be, for example, fragments of the polymers used in the present invention, such as hyaluronic acid, dextran, pullulan, or chondroitin sulfate. While not particularly limited as long as the effects of the present invention are not impaired, sugars may be, for example, cleavage products (fragments) of glycosaminoglycans, i.e., monosaccharides, disaccharides, or oligosaccharides constituting glycosaminoglycans.

[0065] Preferably, saccharide is glucose or hyaluronic acid cleavage product.Therefore, preferably, saccharide of the present invention is glucose, glucuronic acid or N-acetylglucosamine, or disaccharide or oligosaccharide thereof.Preferably, saccharide can be glucuronic acid or its modified compound, or its disaccharide or oligosaccharide.

[0066] The term "cleavage product" as used herein refers to a compound having a smaller molecular weight than the original polymer, which is thought to be obtained when a polymer is subjected to treatment such as hydrolysis, enzymatic treatment, or subcritical treatment. That is, the polymer of the present invention may be a polymer having a viscosity-average molecular weight of more than 3,000 and not more than 500,000, obtained by treating a larger polymeric compound, as described above, and the saccharide of the present invention may be a saccharide having a viscosity-average molecular weight of not more than 3,000, obtained by treating the polymer of the present invention. The cleavage product may be a monomer that is a component of the original polymer, and / or polymers of the monomers with various degrees of polymerization, and / or a mixture thereof.

[0067] "Subcritical processing" refers to contacting a raw material to be extracted with a subcritical fluid (extraction solvent) that has been brought to a subcritical state under predetermined temperature and pressure conditions. For example, water is neither liquid nor gaseous when the pressure is raised to 22.12 MPa or higher and the temperature to 374.15°C or higher. This point is called the critical point of water, and hot water at temperatures and pressures near the critical point is called subcritical water. The hydrolysis action of this subcritical water can be used to obtain desired components from the raw material to be extracted. Conditions for subcritical processing in the present invention include, for example, a temperature of 150°C or higher and 350°C or lower, and the subcritical processing pressure can be set to a value equal to or higher than the saturated vapor pressure at each temperature, e.g., 0.5 MPa or higher and 25 MPa or lower. After subcritical processing, components with molecular weights below a predetermined value are separated and recovered and can be used as the cleavage product in the present invention. Furthermore, there are no particular limitations on the hydrolysis or enzymatic processing, and commonly used reagents and processing methods can be used without any problems.

[0068] The polymer and saccharide of the present invention may be simultaneously obtained by a single subcritical treatment. That is, the polymer and saccharide of the present invention may be subcritically treated products of a polymer compound having a first molecular weight distribution in a molecular weight range of more than 3,000 and not more than 500,000, as measured by viscosity average molecular weight, and a second molecular weight distribution in a molecular weight range of not more than 3,000, as measured by viscosity average molecular weight.

[0069] Salts of the polymer or saccharide of the present invention include metal salts, halogen salts, and sulfate salts. Metal salts are preferably salts of alkali metals or alkaline earth metals. Examples of alkali metals or alkaline earth metals include sodium, potassium, and calcium. Examples of halogens that can be used include chlorine and bromine.

[0070] The cryopreservation solution for blood cells of the present invention contains a polyhydric alcohol. By forming hydrogen bonds with water, polyhydric alcohols can prevent water from freezing and crystallizing, thereby suppressing the destruction of blood cells. The polyhydric alcohol is preferably a polyhydric alcohol having at least three hydroxyl groups. The polyhydric alcohol having at least three hydroxyl groups may be a sugar alcohol. The polyhydric alcohol having at least three hydroxyl groups is preferably an alcohol that is not cytotoxic. Specific examples include glycerol, sorbitol, erythritol, xylitol, diglycerol, triglycerol, and polyglycerol.

[0071] Such polyhydric alcohols having at least three hydroxyl groups penetrate into cells and bind to intracellular water molecules during cell freezing, thereby slowing the rate of ice crystal growth due to intracellular water, and as a result, the polyhydric alcohols having at least three hydroxyl groups can act as cryoprotectants that suppress intracellular ice crystal formation.

[0072] The cryopreservation solution for hematopoietic cells of the present invention is an excellent cryopreservation solution that effectively suppresses ice crystal formation during freezing and recrystallization during thawing, demonstrating a high cryopreservation effect not attainable with conventional techniques. This feature allows the cryopreservation solution of the present invention to significantly reduce damage to cells during cryopreservation and thawing, and allows hematopoietic cells, which have been difficult to freeze while maintaining high viability using conventional cryopreservation solutions, to be cryopreserved stably and with high viability.

[0073] The cryopreservation solution for blood cells of the present invention contains a polymer or a salt thereof at a concentration of approximately 0.1 w / v% or more and 50 w / v% or less. At a concentration lower than 0.1 w / v%, the solvent portion may not be vitrified satisfactorily. At a concentration higher than 50 w / v%, the viscosity may become too high, which may result in poor handling. For example, the concentration of the polymer or a salt thereof is preferably 0.5 w / v% or more. Furthermore, the concentration of the polymer or a salt thereof is preferably 20 w / v% or less. The amount of the polymer or a salt thereof contained in the cryopreservation solution for blood cells of the present invention may be 5 w / v% or more and 20 w / v% or less.

[0074] The concentration of saccharides or their salts in the cryopreservation solution for blood cells of the present invention is approximately 0.1 w / v% or more and 10 w / v% or less. If the concentration of saccharides or their salts is less than 0.1 w / v%, the effects of the present invention may not be fully achieved. Furthermore, if saccharides are added to a concentration of 10 w / v% or more, it is difficult to obtain the additional effect as a cell-protecting component. The weight ratio of polymer to saccharide content in the cryopreservation solution for blood cells of the present invention is desirably polymer:saccharide = 1:1 to 500:1, preferably polymer:saccharide = 1:1 to 50:1, and optimally polymer:saccharide = 1:1 to 20:1.

[0075] The concentration of polyhydric alcohol in the cryopreservation solution for blood cells of the present invention is approximately 1 w / v% or more and 60 w / v% or less. If the polyhydric alcohol concentration is less than 1 w / v%, the effects of the present invention may not be fully achieved. If the polyhydric alcohol concentration is 60 w / v% or more, there is a risk that a good vitrification effect will not be achieved. For example, from the standpoint of handleability, etc., it may be preferable that the polyhydric alcohol concentration be approximately 20 w / v% or less.

[0076] As described above, the use of the cryopreservation solution for hematopoietic cells of the present invention enables cryopreservation of hematopoietic cells while maintaining a high viability. The cryopreservation solution for hematopoietic cells of the present invention exhibits a high cryoprotective effect even on hematopoietic cells that have previously been difficult to freeze while maintaining a high viability. The hematopoietic cells of the present invention include mature blood cells such as leukocytes, granulocytes, lymphocytes, monocytes, and erythrocytes, as well as lineage-restricted progenitor cells and multipotent progenitor cells, such as hematopoietic stem cells, granulocyte-macrophage progenitor cells, megakaryocyte-erythroid progenitor cells, and megakaryocyte progenitor cells, which are at the differentiation stage from hematopoietic stem cells to mature cells.

[0077] The cryopreservation solution of the present invention refers to an aqueous solution prior to addition to a pellet or cell suspension of cells, such as blood cells, to be cryopreserved. Specifically, the cryopreservation solution for blood cells of the present invention contains high-molecular-weight and low-molecular-weight sugars and a polyhydric alcohol in an aqueous solvent. Examples of aqueous solvents include, for example, isotonic solutions in which the salt concentration and sugar concentration are adjusted with sodium ions, potassium ions, calcium ions, etc. to approximately match the osmotic pressure of body fluids or cellular fluids. Specific examples of aqueous solvents include, but are not limited to, water, saline, buffered saline solutions such as phosphate buffered saline (PBS), Dulbecco's phosphate buffered saline, Tris buffered saline (TBS), HEPES-buffered saline, balanced salt solutions such as Hank's balanced salt solution (HBSS), Ringer's solution, lactate Ringer's solution, acetate Ringer's solution, and bicarbonate Ringer's solution. In addition, the solvent may contain other optional components such as an isotonic agent, a chelating agent, a solubilizing agent, a pH adjuster, or an additive that is commonly used as an additive to a cell culture medium, as long as the effects of the present invention are not impaired.

[0078] The cryopreservation solution for hematopoietic cells of the present invention may further contain, as an optional component, a cryoprotective support substance that enhances the cryoprotective effect of the cryopreservation solution. The cryoprotective support substance is a substance other than a sugar or its salt having a viscosity-average molecular weight of 3000 or less. Examples of such substances include amino acids, which are known to form ice nuclei in solution at temperatures higher than the freezing point of intracellular water. Examples of such amino acids include glycine, alanine, valine, asparagine, isoleucine, glutamine, proline, and histidine. Furthermore, the cryoprotective support substance may be a cell membrane-impermeable cryoprotectant, such as a sugar or dextran. Examples of sugars include dextrose, mannose, galactose, fructose, raffinose, lactose, sucrose, maltose, glucose, sorbitol, mannitol, and trehalose. Such a cryoprotective support substance can be contained in the cryopreservation solution of the present invention at a concentration of, for example, about 0.1 w / v % or more and 10 w / v % or less.

[0079] In this specification, the term "optional component" refers to a component that may or may not be included.

[0080] For example, the aqueous solvent of the cryopreservation solution of the present invention may be a 5% glucose aqueous solution. The aqueous solvent may also be a cell culture medium, such as a commercially available medium or a basal medium such as D-MEM, E-MEM, αMEM, RPMI-1640 medium, Ham's F-12, Ham's F-10, or M-199. However, the cryopreservation solution for blood cells of the present invention also includes a case in which predetermined concentrations of polymers, low-molecular-weight sugars, and polyhydric alcohols are added to the culture medium or blood cell suspension after blood cell culture.

[0081] Cryopreservation using the cryopreservation solution for blood cells of the present invention results in minimal damage to the frozen cells. Furthermore, the high cooling rate required for mitigating osmotic shock during cryopreservation, which is required in known vitrification methods, is not required. Therefore, blood cells and other cells can be successfully cryopreserved using a simple, cost-effective method. For example, when using the cryopreservation solution for blood cells of the present invention, blood cells can be successfully frozen to −27°C or below at a cooling rate of, for example, approximately 10°C / min or less. For example, blood cells can be cryopreserved while maintaining a high viability by transferring them to a freezing treatment container or the like along with the cryopreservation solution of the present invention and placing them in a deep freezer at −80°C. No special procedures or equipment are required. The cryopreservation temperature range is not limited as long as it is −27°C or below, but the upper limit is preferably −70°C or below, and preferably −80°C or below. The lower limit is preferably −196°C or above, and preferably −150°C or above.

[0082] The storage period for frozen blood cells in the cryopreservation solution for blood cells of the present invention is not particularly limited, as long as the cryopreserved blood cells maintain properties after thawing that are equivalent to those before freezing, but may be, for example, one week or more, two weeks or more, three weeks or more, four weeks or more, two months or more, three months or more, four months or more, five months or more, six months or more, one year or more, or longer.

[0083] The polymer of the present invention, which has a viscosity-average molecular weight greater than 3,000 and less than 500,000 and contains a monomer with a hydrophilic group as a repeating unit, is a non-permeating cryoprotective reagent and is therefore considered to have low cytotoxicity. Furthermore, in the cryopreservation solution for hematopoietic cells of the present invention, the sugars used together with the polymer function to protect the cells, so the properties of the cells do not change during cryopreservation. Furthermore, the polyhydric alcohol used in the cryopreservation solution for hematopoietic cells of the present invention functions as a cryoprotectant that suppresses intracellular ice crystal formation, so the properties of the cells do not change during cryopreservation. Therefore, by using the cryopreservation solution for hematopoietic cells of the present invention, hematopoietic cells can be cryopreserved stably for long periods of time for regenerative therapy purposes, while maintaining their properties equivalent to those before freezing.

[0084] Thus, by using the cryopreservation solution for hematopoietic cells of the present invention, the vitrification state of the solvent is stabilized in the frozen state, and the toxicity of the cryopreservation solution itself is low, allowing cells to be stably stored in the cryopreservation solution for long periods of time. As used herein, "long-term stable storage" refers to, for example, the following: after thawing hematopoietic cells cryopreserved using the cryopreservation solution for hematopoietic cells of the present invention, the viability of the thawed cells decreases by less than 10%, preferably less than 5%, based on the viability of the cells immediately before storage, after 5 months; after 6 months, by less than 20%, preferably less than 10%, based on the viability of the cells immediately before storage; or after 12 months, by less than 15%, preferably less than 30%. Furthermore, as used herein, "long-term stable storage" refers to, for example, when hematopoietic cells are frozen using the cryopreservation solution for hematopoietic cells of the present invention, stored for a long period of time at -80°C, thawed, and then stored at 4°C, the viability decreases by less than 5% even 24 hours after thawing, based on the viability of the cells immediately after thawing. When cryopreserved using the cryopreservation solution for hematopoietic cells of the present invention, cells are thought to be cryopreserved under conditions with less stress than when DMSO or other cryoprotectants are included. Therefore, by using the cryopreservation solution for hematopoietic cells of the present invention, a very high cell viability can be achieved for cells after freezing and thawing. Furthermore, high cell viability can be achieved not only immediately after thawing, but also for cells that are refrigerated after thawing. The cryopreservation solution for hematopoietic cells of the present invention allows hematopoietic cells to be cryopreserved stably for long periods of time without changing their properties.

[0085] As the polymer of the present invention having a viscosity-average molecular weight of more than 3,000 and not more than 500,000, which contains a monomer having a hydrophilic group as a repeating unit, a polymer that is a biological component is more preferred. By using such a polymer or a salt thereof, cryopreserved blood cells can be thawed, and the thawed blood cells can be administered as is. For example, a preferred polymer of the present invention is hyaluronic acid. In particular, it is hyaluronic acid having a viscosity-average molecular weight of not more than 400,000, preferably not more than 200,000. More particularly, it includes hyaluronic acid having a viscosity-average molecular weight of more than 3,000, more preferably more than 5,000, and not more than 60,000, more preferably not more than 20,000.

[0086] The present invention also relates to a method for cryopreserving blood cells, comprising suspending the blood cells in the cryopreservation solution for blood cells of the present invention and cryopreserving them. By using the cryopreservation solution for blood cells of the present invention, a method for cryopreserving blood cells that improves survival rate and proliferation after thawing can be provided. Because the cryopreservation solution of the present invention does not contain DMSO and causes little cell damage during cryopreservation, the properties of the blood cells can be well maintained during cryopreservation and after thawing. [Example]

[0087] The present invention will be specifically described based on examples, but the present invention is not limited to these.

[0088] <Preparation of cryopreservation solution for testing> (1) In a 2L pressure vessel, high molecular weight hyaluronic acid (manufactured by Shanghai Easier Industrial Development Co., Ltd.) with an average molecular weight of 1 million was mixed with water at a ratio of 20:100, and the mixture was subjected to subcritical treatment at a treatment temperature of 175°C, a treatment pressure of 0.89MPa, and a treatment time of 3 minutes.The subcritical treatment product was then freeze-dried or spray-dried.This resulted in a mixture of high molecular weight hyaluronic acid with an intrinsic viscosity of 0.49dL / g and a viscosity-average molecular weight of 10,000, and low molecular weight hyaluronic acid with an intrinsic viscosity of 0.08dL / g and a viscosity-average molecular weight of 1,000, that is, the high molecular weight and low molecular weight sugars used in the present invention.

[0089] (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, and 9 g of sodium chloride, which have the same composition as Hank's Balanced Salt Solution (HBSS) (Gibco), 10 g of proline, and 100 g of the mixture of high molecular weight and low molecular weight sugars obtained in (1) above, were dissolved to obtain a frozen storage solution for testing (containing 10% by weight of the mixture of high molecular weight and low molecular weight sugars).

[0090] <Cell culture> The human blood cells used were THP-1 cells, a human monocytic cell culture line provided for testing purposes by the RIKEN Cell Bank. THP-1 cells were cultured in RPMI medium (GIBCO) containing 10% bovine serum (Hyclone) at 37°C under 5% CO2 conditions until a sufficient number of cells were obtained.

[0091] <Preparation of cryopreservation solution for blood cells> Example 1 A solution of glycerol (Fujifilm Wako Pure Chemical Industries, Ltd.) suspended in HBSS (Gibco) to a final concentration of 20% was mixed 1:1 with a test cryopreservation solution diluted 5 times with HBSS to prepare a 10:1 diluted cryopreservation solution containing 10% glycerol (containing a 1% by weight mixture of high and low molecular weight sugars. High molecular weight hyaluronic acid with a viscosity average molecular weight of 10,000: low molecular weight hyaluronic acid with a viscosity average molecular weight of 1,000 = 10:1).

[0092] Example 2 A solution of glycerol (Fujifilm Wako Pure Chemical Industries, Ltd.) suspended in HBSS (Gibco) to a final concentration of 20% was mixed 1:1 with the test cryopreservation solution diluted 2-fold with HBSS to prepare a 4-fold diluted cryopreservation solution containing 10% glycerol (containing 2.5% by weight of a mixture of high and low molecular weight sugars. The ratio of high molecular weight hyaluronic acid with a viscosity average molecular weight of 10,000 to low molecular weight hyaluronic acid with a viscosity average molecular weight of 1,000 is 10:1). ).

[0093] Example 3 A solution of glycerol (Fujifilm Wako Pure Chemical Industries, Ltd.) suspended in HBSS (Gibco) to a final concentration of 20% was mixed with the test cryopreservation solution in a 1:1 ratio to prepare a 2x diluted cryopreservation solution containing 10% glycerol (containing a 5% by weight mixture of high and low molecular weight sugars. The ratio of high molecular weight hyaluronic acid with a viscosity average molecular weight of 10,000 to low molecular weight hyaluronic acid with a viscosity average molecular weight of 1,000 is 10:1).

[0094] Test Example 1 The test cryopreservation solution obtained above was used as the cryopreservation solution (containing a 10% mixture of high and low molecular weight sugars. High molecular weight hyaluronic acid with a viscosity average molecular weight of 10,000: low molecular weight hyaluronic acid with a viscosity average molecular weight of 1,000 = 10:1).

[0095] Comparison Example 1 A solution prepared by adding glycerol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to HBSS (Gibco) to a final concentration of 10% was used as the cryopreservation solution of Comparative Example 1.

[0096] Comparative Example 2 A commercially available cryopreservation solution (STEM-CELLBANKER (registered trademark) GMP grade (containing DMSO), manufactured by Zenoac Resources, Inc.; a preservation solution presumably prepared by mixing an aqueous solution in which 5 g of sodium carboxymethylcellulose (molecular weight 760,000) was dissolved in 100 mL of DMSO and 750 mL of distilled water, with an aqueous solution in which 30.0 g of glucose, 0.8 g of sodium bicarbonate, 0.36 g of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and 1.576 g of phosphate buffer were dissolved in 150 mL of distilled water) was used as the cryopreservation solution for Comparative Example 2.

[0097] <Freezing and preserving THP-1 cells using various cryopreservation media> Harvest 1 x 10 THP-1 cells in culture. 6 The cells were suspended in the cryopreservation solutions of Examples 1 to 3, Test Example 1, and Comparative Examples 1 and 2 to a concentration of 100 cells / mL. They were then placed in a cryopreservation container (Mr. Frosty) and frozen at a rate of 1°C / min in a deep freezer at -80°C. The cells were cryopreserved until their next use.

[0098] <Method for evaluating cryopreservation stability> The storage stability of each cryopreservative solution was evaluated by detecting apoptotic cells after freezing and thawing and calculating the ratio of viable cells to the total number of frozen and thawed cells.

[0099] THP-1 cells cryopreserved in each cryopreservation solution were thawed in a 37°C water bath. After replacing the solvent with Flow Cytometry Staining Buffer (R&D Systems, Inc.), viability (membrane damage) and pre-apoptosis were assessed by propidium iodide (PI) staining and Annexin V staining (FITC Annexin V Apoptosis Detection kit, BD Pharmingen). Fluorescence intensity was measured using a flow cytometer (BD FACSCanto II) to identify PI-positive cells (i.e., dead cells) and Annexin V-positive cells (i.e., pre-apoptotic cells). The percentage of PI-positive cells, Annexin V-positive cells, PI / Annexin V-positive cells, and PI / Annexin V-negative cells was then used to calculate the percentage of viable cells among the total cells after thawing. The preservation performance of each cryopreservation solution was then compared.

[0100] The results of detecting stained cells by flow cytometry are shown in Figure 1.

[0101] As shown in Figure 1, the cryopreservation solutions of Examples 1 to 3, which contain glycerol and various proportions of a mixture of high-molecular-weight and low-molecular-weight sugars, showed improved post-thaw cell viability and reduced Annexin V-positive expression, suggesting pre-apoptosis, compared with the cryopreservation solution of Test Example 1, which did not contain glycerol, and the cryopreservation solution of Comparative Example 1, which did not contain a mixture of high-molecular-weight and low-molecular-weight sugars. The post-thaw cell viability also improved as the content of the mixture of high-molecular-weight and low-molecular-weight sugars in the cryopreservation solution increased. Furthermore, the viability improvement effect of Examples 1 to 3 was significantly greater than that of Comparative Example 3, which used a commercially available cryopreservation solution.

[0102] The above results demonstrate that the cryopreservation solution for hematopoietic cells of the present invention has the remarkable effect of enabling cryopreservation of hematopoietic cells with a higher cell viability than commercially available cryopreservation solutions by stably vitrifying the interior of cells, without essentially requiring the addition of cytotoxic cryoprotectants such as DMSO or ethylene glycol, and / or serum or serum-derived proteins, etc. During cryopreservation, the cell membranes and intracellular structures of hematopoietic cells are well protected without damage.

Claims

[Claim 1] In a solvent, 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; Polyhydric alcohol and A cryopreservation solution for blood cells containing:

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