Cryopreservation solution

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] It is known that in the cryopreservation mechanism of cells, 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 having a viscosity-average molecular weight greater than 3000 and less than or equal to 500000 and containing a monomer having a hydrophilic group as a repeating unit, or a salt thereof, and a saccharide having a viscosity-average molecular weight of 3000 or less or a salt thereof.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Summary of the Invention

Problems 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 cell properties without using substances such as dimethyl sulfoxide, which have been widely used as cryoprotective substances but are known to have cytotoxicity and differentiation-inducing properties. However, depending on the type of biological sample, a sufficient cryopreservation effect may not be obtained.

[0009] In particular, in the case of cryopreservation of hematopoietic cells as biological samples, the cell survival rate after thawing may decrease compared to cryopreservation of mesenchymal stem cells and the like. Therefore, there is a need to develop a cryopreservation solution that can provide a high cryopreservation effect for such hematopoietic cells.

[0010] The present invention has been made in view of the above problems, and provides a cryopreservation solution for appropriately cryopreserving hematopoietic cells. In particular, an object of the present invention is to provide a highly biocompatible cryopreservation solution for hematopoietic cells that can stably cryopreserve hematopoietic cells while maintaining a high cell survival rate without adding cryoprotective substances having cytotoxicity and differentiation-inducing properties such as dimethyl sulfoxide.

Means for Solving the Problems

[0011] The present invention relates to a cryopreservation solution for hemocyte cells, which contains, in a solvent, a polymer having a viscosity average molecular weight greater than 3000 and less than or equal to 500000, 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 3000 or less or a salt thereof, and a polyhydric alcohol. The salt of the polymer or saccharide is preferably a metal salt, a halogen salt or a sulfate salt. As the metal salt, a salt of an alkali metal or an alkaline earth metal is preferred. As the alkali metal or alkaline earth metal, sodium, potassium, calcium, etc. are selected. As the halogen, chlorine, bromine, etc. can be used.

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

[0013] In the present invention, it is desirable that the polymer having a viscosity average molecular weight greater than 3000 and less than or equal to 500000, the polymer containing a monomer having a hydrophilic group as a repeating unit or a salt thereof, be included as a main component, and the saccharide having a viscosity average molecular weight of 3000 or less or a salt thereof be included as a sub-component. In this specification, the main component refers to the component with the highest weight ratio among the components dissolved in the solvent. Constituent components other than the main component are sub-components.

[0014] In the polymer containing a monomer having a hydrophilic group as a repeating unit used in the present invention, it is desirable that the hydrophilic group is not modified or, even if it is modified, the number of the hydrophilic groups is 50% or less of the total number of the hydrophilic groups. It is presumed that the hydrophilic groups in the polymer are involved in protecting the cells to be frozen simultaneously with the vitrification of the solvent and the substitution of the sugar with a molecular weight of 3000 or less and the water around the biological sample. If the hydrophilic group is modified and becomes hydrophobic, the cryopreservation effect of the cells will decrease. Therefore, it is desirable to exclude a polymer such as carboxypolyamino acid as the main component.

[0015] The cryopreservation solution for hematopoietic cells of the present invention preferably does not contain dimethyl sulfoxide, which is a cryoprotective substance that can function as a differentiation factor. Further, the cryopreservation solution for hematopoietic cells of the present invention preferably does not contain cryoprotective substances with cytotoxicity such as ethylene glycol. This is because these are harmful to the 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. This is because the viscosity can be adjusted low, making it easy to handle as a cryopreservation solution.

[0017] A cryopreservation solution for hematopoietic cells, which is a monomer having a hydrophilic group and is a monomer having at least one hydrophilic group selected from the group consisting of a hydroxyl group and a carboxylic acid group and its salt, is preferred.

[0018] A cryopreservation solution for hematopoietic cells, in which the polymer further contains a nitrogen-containing monomer having an optionally substituted amino group or an optionally substituted amide group as a repeating unit, is preferred.

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

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

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

[0022] A cryopreservation solution for hematopoietic cells, in which the polymer is a polymer containing a plurality of sugar residues, is preferred.

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

[0024] A cryopreservation solution for hematopoietic cells in which the saccharide is glucose, fructose, galactose, or uronic acid obtained by oxidizing the alcohol group thereof or 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 solution for hematopoietic cells in which the saccharide is glucose, glucuronic acid, or N-acetylglucosamine is preferred.

[0026] A cryopreservation solution for hematopoietic cells in which the polyhydric alcohol is a polyhydric alcohol having at least three hydroxyl groups is preferred.

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

[0028] A cryopreservation solution for hematopoietic cells in which the concentration of the saccharide or its salt in the cryopreservation solution is 0.1 w / v% or more and 10 w / v% or less is preferred.

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

[0030] A cryopreservation solution for hematopoietic cells in which the hematopoietic cells are megakaryocyte progenitor cells is preferred.

[0031] A cryopreservation solution for hematopoietic cells in which the hematopoietic cells are granulocytes, lymphocytes, monocytes, or erythrocytes is preferred.

[0032] A cryopreservation solution for hematopoietic cells in which the hematopoietic cells are leukocytes is preferred.

[0033] The present invention also relates to a method for cryopreserving hematopoietic cells, which comprises suspending hematopoietic cells in any of the above 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: TIFF0007716851000001.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. TIFF0007716851000002.tif3559The viscosity average molecular weight M can be calculated using the above Mark Hoeing-Sakurada equation, the intrinsic viscosity derived from the measurement, and the values of K and α published in literature.

[0037] K and α are numerical values that vary depending on the type of polymer. For example, the values of K and α are disclosed in a number of published documents such as "Polymer Materials Handbook" (edited by the Society of Polymer Science, Japan), and the viscosity-average molecular weight can be calculated using the published values.

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

[0039] In the case of monosaccharides, disaccharides, and compounds considered to be single molecules, the molecular weight can be clearly specified from the structural formula. Therefore, in the present invention, the molecular weight specified from the structural formula is fictitiously treated as the viscosity-average molecular weight.

[0040] As the solvent used for preparing the cryopreservation solution of the present invention, it is desirable to use an aqueous solvent such as water. In particular, it is preferably an isotonic solution in which the salt concentration, sugar concentration, etc. are adjusted by sodium ions, potassium ions, calcium ions, etc. so as to be substantially the same as the osmotic pressure of body fluids and cell fluids. Specifically, for example, water, physiological saline, phosphate buffered saline (PBS) which is physiological saline having a buffering effect, Dulbecco's phosphate buffered saline, Tris Buffered Saline (TBS), HEPES buffered saline, etc., balanced salt solutions such as Hank's balanced salt solution, Ringer's solution, lactate Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution, or basal media for animal cell culture such as D-MEM, E-MEM, αMEM, RPMI-1640 medium, Ham's F-12, Ham's F-10, M-199, and other commercially available media can be mentioned.

[0041] In addition, the solvent may contain calcium chloride, magnesium chloride, magnesium sulfate, potassium chloride, potassium dihydrogen phosphate, sodium bicarbonate, disodium hydrogen phosphate, glucose, sodium chloride, amino acids, etc. Proline is preferably selected as the amino acid.

Advantages of the Invention

[0042] The cryopreservation solution for hematopoietic cells of the present invention has a viscosity average molecular weight exceeding 3000 and not exceeding 500000, which imparts vitrification performance by a high molecular weight component, inhibits ice crystal formation and growth near the cell membrane by low molecular weight saccharides with a viscosity average molecular weight of 3000 or less, and has a protective effect on cells, and the anti-freezing function of polyhydric alcohols that are intracellularly permeable and replace water molecules inside the cells. In hematopoietic cells, it can prevent damage to the cell membrane during cryopreservation and bring about a high survival rate and proliferation rate after thawing applicable to advanced cell therapy. The cryopreservation solution for hematopoietic cells of the present invention can show a high cryopreservation effect on many types of cells including nucleated cells and non-nucleated cells, including those for hematopoietic cells, which was difficult with the prior art, without using substances with high cytotoxicity.

[0043] That is, in the cryopreservation solution for hematopoietic cells of the present invention, since substances with high cytotoxicity such as DMSO are not contained, adverse effects on cells are suppressed, and the survival rate and quality of cells during and after cryopreservation can be maintained. It is possible to add chemical substances that may have cytotoxicity such as DMSO and ethylene glycol at low concentrations that do not impair the function of the cells.

[0044] In addition, since the cryopreservation solution for hematopoietic cells of the present invention does not contain serum and / or serum-derived proteins, the cells to be frozen are not contaminated with bacteria or viruses. It is possible to add proteins that are not contaminated with bacteria or viruses.

[0045] In addition, in the cryopreservation of cells using the cryopreservation solution for hematopoietic cells of the present invention, a large cooling rate for cell protection is not required during cooling, and a low temperature using liquid nitrogen or the like is not required either. Therefore, the cryopreservation operation is simple and the cost efficiency is good.

Brief Description of the Drawings

[0046]

Figure 1

Modes for Carrying Out the Invention

[0047] The cryopreservation solution for hematopoietic cells of the present invention is a cryopreservation solution for hematopoietic cells containing a polymer having a viscosity average molecular weight greater than 3000 and not exceeding 500000 or a salt thereof, a saccharide having a viscosity average molecular weight not exceeding 3000 or a salt thereof, and a polyhydric alcohol in a solvent. 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 its salt of the present invention is desirably not exceeding 400000, particularly not exceeding 200000. This is because the viscosity can be adjusted low and it 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 from the following method and calculation formula.

[0050] The following describes the method for measuring the intrinsic viscosity and the method for calculating the viscosity average molecular weight using the intrinsic viscosity. 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) Dissolve the polymer or saccharide sample in a standard solution at 30 °C to prepare a stock solution. When the polymer or saccharide sample is obtained as a solution, the solid content obtained by removing the solvent from the solution is used as the polymer or saccharide sample. In the case of a mixed sample of a polymer and a saccharide, a mixed sample containing a plurality of polymers, or a mixed sample containing a plurality of saccharides, after separating and fractionating each substance, the one obtained by removing the solvent from each substance is used as the polymer or saccharide sample. Also, when the polymer and / or saccharide is unknown, identify the substance by HPLC, LC-MS, LC-IR, etc. for the polymer and / or saccharide. When there are a plurality of unknown polymers and / or saccharides, separate and fractionate each component, identify the substance by HPLC, LC-MS, LC-IR, etc. for each polymer and / or saccharide, and calculate the viscosity average molecular weight as described below. Note that even if it is a mixture containing impurities in the polymer or saccharide, if there is no influence on the viscosity (for example, impurities such as metal salts), the mixture is used as the polymer or saccharide sample. Also, when it contains impurities that affect the calculation of the viscosity average molecular weight, remove the impurities or measure after fractionating the polymer or saccharide. Measure the viscosity of each of the standard solution and the stock solution, and adjust so that the relative viscosity of the stock solution with respect to the standard solution is 2.0 to 2.4. (3) Dilute the stock solution at 30 °C to 5 / 4, 5 / 3, and 5 / 2 times using the standard solution at 30 °C, respectively. (4) Measure the viscosities of the standard solution, stock solution, and diluted solutions at 30 °C, respectively. Use an E-type viscometer for viscosity measurement. (5) Divide the viscosity of each of the stock solution and the diluted solutions by the viscosity of the standard solution to obtain the relative viscosity (η r ), and derive the reduced viscosity based on the following formula. TIFF0007716851000003.tif2146 Here, η sp : Reduced viscosity of the polymer or saccharide [mL / g], η r : Relative viscosity of the polymer or saccharide [-], C: Concentration of the polymer or saccharide [g / mL]. (6) Plot the relationship between the concentration of the polymer or saccharide and the reduced viscosity of the polymer or saccharide, respectively, and draw an approximate straight line. The value of the intercept (polymer or saccharide concentration = 0) of the approximate straight line is taken as the limiting viscosity. Viscosity average molecular weight: The viscosity-average molecular weight is calculated from the intrinsic viscosity. TIFF0007716851000004.tif3559The viscosity-average molecular weight M is determined from the intrinsic viscosity derived by measurement and the values of K and α published in the literature and the like in the above-mentioned Mark-Houwink Sakurada equation. In the case of hyaluronic acid, K = 3.6×10 -4 and α = 0.78 are substituted to determine the viscosity-average molecular weight M. For pullulan and gelatin used in the examples, K = 9×10 from the literature values -4 , α = 0.5, and for dextran, K = 6.3×10 -8 , α = 1.4, for chondroitin sulfate, K = 5.8×10 -4 , α = 0.74, and for carboxypolylysine, K = 2.78×10 -5 , α = 0.87 are used.

[0051] K and α are values that vary depending on the type of polymer. For example, the values of K and α are disclosed in many published documents such as "Polymer Materials Handbook" (edited by the Society of Polymer Science, Japan), and the viscosity-average molecular weight is calculated using the published values.

[0052] In the present invention, the molecular weight calculated by this method is taken as the viscosity-average molecular weight. In the case of monosaccharides and disaccharides such as sucrose and glucuronic acid, and compounds considered to be single molecules, since the molecular weight is clearly specified from the structural formula, the molecular weight specified from the structural formula is fictitiously treated as the viscosity-average molecular weight.

[0053] In the cryopreservation using the cryopreservation solution of the present invention for hematopoietic cells, in the cooling process of the sample to be cryopreserved, water molecules of the solvent are trapped in the matrix formed by the polymer chains. Since the polymer chains contain hydrophilic groups, the molecular motion of water in the solvent is restricted during cooling, and the water can be solidified and / or frozen in a vitrified state without crystallization. Thus, due to the action of the polymer chains, the inside of the cells is dehydrated and vitrified. Therefore, in the cryopreservation using the cryopreservation solution of the present invention for hematopoietic cells, it is not necessary to increase the concentration of the solute (cryoprotectant) or increase the cooling rate as in the conventional vitrification method. In the present invention, since the formation of ice crystals inside the cells is suppressed by the action of the polymer chains, the osmotic shock in the cells during freezing, which has been a problem in the conventional vitrification method of dehydrating the inside of the cells using the osmotic pressure difference generated inside and outside the cells and vitrifying the inside of the cells, can be reduced. Also, since recrystallization does not occur during the dissolution of the frozen cells, it is considered that the damage to the cells due to thawing is small.

[0054] The viscosity average molecular weight of the polymer of the present invention exceeds 3000 and is 500000 or less. With a viscosity average molecular weight of this degree, the glassy state, which is amorphous in the frozen state, can be stabilized. The cells are less likely to be damaged by cooling and freezing, and the cells can be stably cryopreserved. Therefore, the survival rate of the cells including hematopoietic cells after thawing the biological sample after cryopreservation is high. When the viscosity average molecular weight of the polymer is 3000 or less, vitrification may not occur well. Also, when the viscosity average molecular weight of the polymer is greater than 500000, problems may occur such as a significant increase in viscosity, a decrease in solubility, or the prepared solution foaming and the handling property deteriorating. The viscosity average molecular weight is preferably, for example, 5000 or more. Also, a polymer having a viscosity average molecular weight of 400000 or less, more preferably 200000 or less, and particularly preferably 150000 or less is preferred because the viscosity can be adjusted low and it is easy to handle as a cryopreservation solution.

[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, examples of monosaccharides substituted with a carboxyl group include uronic acids. 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 is a polymer containing the above-described monosaccharides as repeating units. For example, the polymer of the present invention can be a polymer containing a pentose, hexose, or uronic acid, which may be substituted, or a combination thereof, as a repeating unit. Further, the polymer of the present invention may 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 can be a glycosaminoglycan. Further, it may be a sulfated polysaccharide in which one or more hydroxyl groups are substituted with a sulfooxy group. Although not limited thereto, examples of the polymer of the present invention include hyaluronic acid, dextran, pullulan, or chondroitin sulfate.

[0060] The polymers used in the present invention may be of natural origin, chemically synthesized, or commercially available polymers. Natural or commercially available polymer compounds with a larger molecular weight may be subjected to treatments such as hydrolysis, enzymatic treatment, or subcritical treatment to obtain their cleavage products, and the molecular weight may be adjusted to obtain the polymers of the present invention. Each monomer may also be of natural origin, may be used after modifying and substituting natural monomers, or may be chemically synthesized. For example, preferably, the monomers contained in the polymers of the present invention are biological components. It is considered that the cryopreservation solution containing the polymer has low cytotoxicity.

[0061] It is desirable that the hydrophilic groups of the polymers of the present invention are either unmodified or, if modified, 50% or less of the total number of hydrophilic groups, that is, either no substituents are introduced into the polymer chain or, if introduced, 50% or less of the total number of hydrophilic groups. Since the hydrophilic groups of the polymer, particularly OH groups, NH2 groups, and COOH groups, are presumed to contribute to the protection of frozen cells, the vitrification of the solvent, and the substitution of sugars and water around the cells, it is considered advantageous for improving the survival rate of cells after thawing that these functional groups are unmodified.

[0062] Also, it is considered that the hydrophilic groups of the polymers of the present invention hold low-molecular-weight saccharides by hydrogen bonds. The presence of such polymers holding low-molecular-weight saccharides around biological samples such as cells is presumed to promote the substitution of water molecules and saccharides near the cell membrane. Therefore, if the hydrophilic groups are modified, the effect of holding low-molecular-weight saccharides by the hydrophilic groups will decrease, and even if low-molecular-weight saccharides coexist, there is a risk that they will not sufficiently contribute to improving the survival rate of cells. Therefore, it may not be preferable to modify OH groups or NH2 groups with carboxylic acids or the like.

[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 "cleavage product" used in the present invention means a compound having a molecular weight smaller than that of the original polymer, which is considered to be obtained when the polymer is subjected to treatments such as hydrolysis, enzymatic treatment, and subcritical treatment. That is, the polymer of the present invention may be a polymer having a viscosity-average molecular weight of more than 3000 and 500000 or less, which is obtained by treating a larger polymer compound as described above. The saccharide of the present invention may be a saccharide having a viscosity-average molecular weight of 3000 or less, which is obtained by treating the polymer of the present invention. The cleavage product can be a monomer that is a constituent component of the original polymer and / or a polymer having various degrees of polymerization of the monomer and / or a mixture thereof.

[0067] "Subcritical treatment" means bringing a subcritical fluid as an extraction solvent in a subcritical state under conditions of a predetermined temperature and a predetermined pressure into contact with a raw material to be extracted. For example, when water is heated to a pressure of 22.12 MPa or more and a temperature of 374.15 °C or more, it shows a state that is neither liquid nor gas. This point is called the critical point of water, and hot water at a temperature and pressure in the vicinity lower than the critical point is called subcritical water. By using the hydrolysis action of this subcritical water, a desired component can be obtained from the raw material to be extracted. As the conditions for subcritical treatment in the present invention, for example, the temperature is 150 °C or more and 350 °C or less, and the subcritical treatment pressure can be set to be equal to or higher than the saturated vapor pressure at each temperature. For example, it can be 0.5 MPa or more and 25 MPa or less. After subcritical treatment, components having a molecular weight below a predetermined value are separated and recovered and can be used as the cleavage product in the present invention. Also, hydrolysis and enzymatic treatment are not particularly limited, and reagents and treatment methods commonly used can be used without problems.

[0068] The polymer and saccharide of the present invention may be obtained simultaneously by a single subcritical treatment. That is, the polymer and saccharide in 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 500000 or less as the viscosity-average molecular weight and a second molecular weight distribution in a molecular weight range of 3000 or less as the viscosity-average molecular weight.

[0069] Examples of the salts of the polymer or saccharide of the present invention include metal salts, halogen salts, and sulfates. As the metal salt, a salt of an alkali metal or an alkaline earth metal is desirable. As the alkali metal or alkaline earth metal, sodium, potassium, calcium, etc. are selected. As the halogen, chlorine, bromine, etc. can be used.

[0070] The cryopreservation solution for blood cell - based cells of the present invention contains a polyhydric alcohol. The polyhydric alcohol forms a hydrogen bond with water, thereby inhibiting water from freezing and crystallizing, and can suppress the destruction of blood cells. The polyhydric alcohol is preferably a polyhydric alcohol having at least 3 hydroxyl groups. The polyhydric alcohol having at least 3 hydroxyl groups may be a sugar alcohol. As the polyhydric alcohol having at least 3 hydroxyl groups, an alcohol having no cytotoxicity is preferable. Specifically, for example, glycerol, sorbitol, erythritol, xylitol, diglycerol, triglycerol, polyglycerol, etc. can be mentioned.

[0071] Such a polyhydric alcohol having at least 3 hydroxyl groups penetrates into the cell during cell freezing and binds to the water molecules inside the cell. Thereby, it slows down the growth rate of ice crystals formed by the water inside the cell. As a result, the polyhydric alcohol having at least 3 hydroxyl groups can act as a cryoprotectant that suppresses the formation of ice crystals inside the cell.

[0072] The cryopreservation solution for hematopoietic cells of the present invention can trap water molecules with polymer chains during freezing, prevent ice crystals from forming in the solvent part, and vitrify, thereby suppressing the rupture of biological samples such as cells due to ice crystal formation. By replacing water molecules near the boundary tissue between the biological sample such as the cell membrane and the solvent, it inhibits the formation and growth of ice crystals near the cell membrane and protects the cell membrane. A combination with a polyhydric alcohol, which is a cryoprotectant that suppresses the formation of ice crystals inside the cell, can effectively suppress ice crystal formation during freezing and recrystallization during thawing of the cells, and is an excellent cryopreservation solution showing a high cryopreservation effect that could not be obtained in the prior art. Due to this feature, the cryopreservation solution of the present invention can significantly reduce the damage to cells during cryopreservation and thawing, and hematopoietic cells that were difficult to freeze while maintaining a high survival rate with conventional cryopreservation solutions can also be cryopreserved while maintaining a stable and high survival rate.

[0073] The cryopreservation solution for hematopoietic cells of the present invention contains a polymer or a salt thereof at a concentration of about 0.1 w / v% or more and 50 w / v% or less. If the concentration is lower than 0.1 w / v%, the solvent part may not be vitrified well. Also, at a concentration higher than 50 w / v%, the viscosity may become too high, which may deteriorate the handling property. For example, the concentration of the polymer or a salt thereof is preferably 0.5 w / v% or more. Also, 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 hematopoietic cells of the present invention may be 5 w / v% or more and 20 w / v% or less.

[0074] The concentration of the saccharide or its salt in the cryopreservation solution for hematopoietic cells of the present invention is about 0.1 w / v% or more and 10 w / v% or less. If the concentration of the saccharide or its salt is less than 0.1 w / v%, the effects of the present invention may not be fully obtained. Further, when the saccharide is added at a concentration of 10 w / v% or more, it is difficult to obtain further effects as a cell protection component. The weight ratio of the content of the polymer to the saccharide in the cryopreservation solution for hematopoietic cells of the present invention is preferably polymer:saccharide = 1:1 to 500:1, more preferably polymer:saccharide = 1:1 to 50:1, and most preferably polymer:saccharide = 1:1 to 20:1.

[0075] The concentration of the polyhydric alcohol in the cryopreservation solution for hematopoietic cells of the present invention is about 1 w / v% or more and 60 w / v% or less. If the concentration of the polyhydric alcohol is less than 1 w / v%, the effects of the present invention may not be fully obtained. If the concentration of the polyhydric alcohol is 60 w / v% or more, there is a risk that a good vitrification effect cannot be obtained. For example, from the viewpoint of handling properties and the like, the concentration of the polyhydric alcohol may preferably be about 20 w / v% or less.

[0076] As described above, by using the cryopreservation solution for hematopoietic cells of the present invention, hematopoietic cells can be cryopreserved while maintaining a high survival rate. The cryopreservation solution for hematopoietic cells of the present invention exhibits a high cryoprotective effect even on hematopoietic cells that were difficult to cryopreserve while maintaining a high survival rate. The hematopoietic cells of the present invention include mature blood cells such as white blood cells, granulocytes, lymphocytes, monocytes, and red blood cells, as well as lineage-restricted progenitor cells and pluripotent progenitor cells such as hematopoietic stem cells, granulocyte-macrophage progenitor cells, megakaryocyte-erythroblast progenitor cells, and megakaryocyte progenitor cells, which are differentiation stages from hematopoietic stem cells to mature cells.

[0077] The cryopreservation solution in the present invention refers to an aqueous solution before being added to a pellet or cell suspension of cells such as blood cell-derived cells to be cryopreserved. That is, the cryopreservation solution for blood cell-derived cells of the present invention contains a polymer, low molecular weight saccharides, and polyhydric alcohols in an aqueous solvent. As the aqueous solvent, for example, an isotonic solution in which the salt concentration, sugar concentration, etc. are adjusted by sodium ions, potassium ions, calcium ions, etc. so as to be almost the same as the osmotic pressure of body fluid or cell fluid may be used. Specifically, for example, water, physiological saline, phosphate buffered saline (PBS) which is a physiological saline having a buffering effect, Dulbecco's phosphate buffered saline, Tris Buffered Saline (TBS), HEPES buffered saline, etc., balanced salt solutions such as Hank's balanced salt solution (HBSS), Ringer's solution, lactate Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution, etc. are exemplified, but are not limited thereto. Further, as long as the effects of the present invention are not impaired, the solvent may contain other optional components such as, for example, tonicity agents, chelating agents, solubilizing aids, pH adjusters, and additives commonly used as additives to cell culture media.

[0078] In addition, the cryopreservation solution for hematopoietic cells of the present invention may further contain, as other optional components, a cryoprotective support substance that assists the cryoprotective action of the cryopreservation solution. The cryoprotective support substance is a substance different from saccharides or their salts having a viscosity average molecular weight of 3000 or less. Examples of such substances include amino acids, which are known to be substances that form ice nuclei in a solution at a temperature higher than the freezing of intracellular water. Examples of such amino acids include glycine, alanine, valine, asparagine, isoleucine, glutamine, proline, and histidine. Further, as the cryoprotective support substance, a cell membrane non-permeable cryoprotective substance may be used, and examples thereof include saccharides and dextran. Examples of saccharides include dextrose, mannose, galactose, fructose, raffinose, lactose, sucrose, maltose, glucose, sorbitol, mannitol, and trehalose. Such a cryoprotective support substance may be contained in the cryopreservation solution of the present invention at a concentration of, for example, 0.1 w / v% or more and 10 w / v% or less.

[0079] In the present specification, "optional component" means a component that may or may not be contained.

[0080] For example, the aqueous solvent of the cryopreservation solution of the present invention may be a 5% glucose aqueous solution or the like. The aqueous solvent may also be, for example, a commercially available medium or a medium for cell culture such as a basal medium such as D-MEM, E-MEM, αMEM, RPMI-1640 medium, Ham's F-12, Ham's F-10, and M-199. However, the cryopreservation solution for hematopoietic cells of the present invention also includes cases where a polymer, a low molecular weight saccharide, and a polyhydric alcohol at a predetermined concentration are added to the culture solution after culturing hematopoietic cells or the suspension of hematopoietic cells and used.

[0081] In the cryopreservation of hematopoietic cells using the cryopreservation solution of the present invention, the damage to the cells to be frozen is low. Also, a large cooling rate required for reducing the osmotic shock during cryopreservation required by the known vitrification method is not necessary. Therefore, hematopoietic cells and the like can be favorably cryopreserved by a simple and cost-effective method. For example, when the cryopreservation solution for hematopoietic cells of the present invention is used, the hematopoietic cells are favorably frozen by cooling to -27°C or lower, for example, at a cooling rate of about 10°C / min or lower. For hematopoietic cells, for example, together with the cryopreservation solution of the present invention, they are transferred to a cryopreservation container or the like and left in a -80°C deep freezer, thereby being cryopreserved while maintaining a high survival rate. No special techniques or instruments are required. The range of the cryopreservation temperature is not limited as long as it is -27°C or lower, but as an upper limit, it is preferably -70°C or lower, more preferably -80°C or lower. Also, as a lower limit, it is preferably -196°C or higher, more preferably -150°C or higher.

[0082] The storage period of the cryopreserved hematopoietic cells using the cryopreservation solution for hematopoietic cells of the present invention is not particularly limited as long as the cryopreserved hematopoietic cells retain the same properties as before freezing after thawing. For example, it can be 1 week or longer, 2 weeks or longer, 3 weeks or longer, 4 weeks or longer, 2 months or longer, 3 months or longer, 4 months or longer, 5 months or longer, 6 months or longer, 1 year or longer, or longer.

[0083] The polymer of the present invention having a viscosity average molecular weight greater than 3,000 and not exceeding 500,000, which contains a monomer having a hydrophilic group as a repeating unit, is a non-permeating cryoprotective reagent and is considered to have low cytotoxicity. Further, in the cryopreservation solution for hematopoietic cells of the present invention, since the saccharide used together with the polymer functions for cell protection, the properties of the cells do not change during cryopreservation. Furthermore, since the polyhydric alcohol used in the cryopreservation solution for hematopoietic cells of the present invention functions as a cryoprotectant that suppresses ice crystal formation in cells, no change in the properties of the cells occurs during cryopreservation. Therefore, by using the cryopreservation solution for hematopoietic cells of the present invention, hematopoietic cells can be cryopreserved stably and for a long period for the purpose of regenerative medicine while maintaining their properties equivalent to those before freezing.

[0084] Thus, by using the cryopreservation solution for hematopoietic cells of the present invention, the vitrified state of the solvent is stabilized in the frozen state, and the toxicity of the cryopreservation solution itself is low. Therefore, cells can be stably stored in the cryopreservation solution for a long time. In this specification, long-term stable storage means, for example, that the survival rate of thawed cells after thawing hematopoietic cells cryopreserved using the cryopreservation solution for hematopoietic cells of the present invention is less than about 10%, preferably less than about 5% after 5 months, or less than about 20%, preferably less than about 10% after 6 months, or less than about 15%, preferably less than about 30% after 12 months, based on the survival rate of the cells immediately before storage. Also, in this specification, long-term stable storage means, for example, when hematopoietic cells are frozen using the cryopreservation solution for hematopoietic cells of the present invention and stored at -80°C for a long time, then the cells are thawed, and subsequently the thawed cells are stored at 4°C, it means that even 24 hours after thawing, only a decrease in the survival rate of less than 5% is observed based on the cell survival rate immediately after thawing. In the cryopreservation using the cryopreservation solution for hematopoietic cells of the present invention, it is considered that the cells are cryopreserved under conditions with less stress compared to the case where a cryoprotective substance such as DMSO is included. Therefore, by using the cryopreservation solution for hematopoietic cells of the present invention, a very high cell survival rate can be obtained for the cells after freezing and thawing. Furthermore, not only immediately after thawing, but also cells stored refrigerated after thawing can exhibit a high cell survival rate. The cryopreservation solution for hematopoietic cells of the present invention can stably cryopreserve hematopoietic cells for a long time without changing the properties of the cells.

[0085] The polymer of the present invention having a viscosity average molecular weight greater than 3000 and not more than 500000, which contains a monomer having a hydrophilic group as a repeating unit, is preferably, for example, a polymer that is a biological component. By using such a polymer or a salt thereof, cryopreserved blood cell-based cells can be thawed, and the thawed blood cell-based cells can be administered as they are. 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 400000, desirably not more than 200000. More particularly, hyaluronic acid having a viscosity average molecular weight greater than 3000, more desirably greater than 5000, and not more than 60000, more desirably not more than 20000 can be mentioned.

[0086] The present invention also relates to a method for cryopreserving blood cell-based cells, which comprises suspending blood cell-based cells in the cryopreservation solution for blood cell-based cells of the present invention and cryopreserving them. By using the cryopreservation solution for blood cell-based cells of the present invention, a method for cryopreserving blood cell-based cells with good post-thaw survival rate and proliferation can be provided. The cryopreservation solution of the present invention does not contain DMSO and causes less cell damage during cryopreservation, so the properties of blood cell-based cells can be maintained well during and after cryopreservation.

Examples

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

[0088] <Preparation of Test Cryopreservation Solution> (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 in which glycerol (manufactured by Fujifilm Wako Pure Chemical Corporation) was suspended in HBSS (Gibco) to a final concentration of 20% and a test cryopreservation solution diluted 5-fold with HBSS were mixed at a ratio of 1:1 to prepare a 10-fold diluted cryopreservation solution containing 10% glycerol (containing a mixture of 1 wt% high-molecular-weight and low-molecular-weight saccharides. 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 in which glycerol (manufactured by Fujifilm Wako Pure Chemical Corporation) was suspended in HBSS (Gibco) to a final concentration of 20% and a test cryopreservation solution diluted 2-fold with HBSS were mixed at a ratio of 1:1 to prepare a 4-fold diluted cryopreservation solution containing 10% glycerol (containing a mixture of 2.5 wt% high-molecular-weight and low-molecular-weight saccharides. 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). )

[0093] · Example 3 A solution in which glycerol (manufactured by Fujifilm Wako Pure Chemical Corporation) was suspended in HBSS (Gibco) to a final concentration of 20% and the test cryopreservation solution were mixed at a ratio of 1:1 to prepare a 2-fold diluted cryopreservation solution containing 10% glycerol (containing a mixture of 5 wt% high-molecular-weight and low-molecular-weight saccharides. 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).

[0094] · Test Example 1 The test cryopreservation solution obtained above was used as the cryopreservation solution (containing a mixture of 10% high-molecular-weight and low-molecular-weight saccharides. 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] · Comparative Example 1 A solution in which glycerol (manufactured by Fujifilm Wako Pure Chemical Corporation) was added 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 Xenoa Resource Co., Ltd., a solution prepared by dissolving 5 g of sodium carboxymethylcellulose (molecular weight 760,000) in 100 mL of DMSO and 750 mL of distilled water, and a solution prepared by dissolving 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 in 150 mL of distilled water, and the two solutions were mixed) was used as the cryopreservation solution for Comparative Example 2.

[0097] <Cryopreservation of THP-1 cells using each cryopreservation solution> THP-1 cells in culture were collected and suspended in the cryopreservation solutions of Examples 1 to 3, Test Example 1, and Comparative Examples 1 and 2 at a concentration of 1×10 6 cells / mL. Then, they were placed in a cryopreservation container (Mr. Frosty) and frozen at a freezing rate of 1°C / min in a -80°C deep freezer. The cells were cryopreserved until use.

[0098] <Evaluation method for cryopreservation stability> The storage stability of each cryopreservation solution was evaluated by detecting apoptotic cells after freeze-thawing and calculating the ratio of viable cells to the total number of freeze-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 analyzed by performing Propidium Iodide (PI) staining and Annexin V staining (FITC Annexin V Apoptosis Detection kit, BD pharmingen), and measuring fluorescence intensity by flow cytometry (BD FACSCanto II) to analyze PI-positive cells (i.e., dead cells) and Annexin V-positive cells (i.e., pre-apoptotic cells). Then, the proportion of viable cells among all the cells after thawing was calculated based on the proportions of PI-positive cells, Annexin V-positive cells, PI / Annexin V-positive cells, and PI / Annexin V-negative cells, and the storage performance of each cryopreservation solution was compared.

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

[0101] As shown in Figure 1, compared with the cryopreservation solution of Test Example 1 that does not contain glycerol and the cryopreservation solution of Comparative Example 1 that does not contain a mixture of high-molecular-weight and low-molecular-weight saccharides, by using the cryopreservation solutions of Examples 1 to 3 that contain glycerol and a mixture of high-molecular-weight and low-molecular-weight saccharides in various ratios, an improvement in the viability of cells after thawing and a decrease in the expression of Annexin V positivity, which indicates pre-apoptosis, were observed. As the content of the mixture of high-molecular-weight and low-molecular-weight saccharides in the cryopreservation solution increased, the cell viability after thawing was further improved. Also, the viability improvement effect of Examples 1 to 3 was significantly more effective than that of Comparative Example 3, which is a commercially available cryopreservation solution.

[0102] From the above results, it can be seen that the cryopreservation solution for hematopoietic cells of the present invention has a remarkable effect that it can cryopreserve hematopoietic cells with a higher cell survival rate than commercially available cryopreservation solutions, basically without the need to add cytotoxic cryoprotective substances such as DMSO and ethylene glycol, and / or serum and serum-derived proteins, etc., by stably vitrifying the inside of the cells. During cryopreservation, the cell membrane and intracellular structure of hematopoietic cells are well protected without being damaged.

Claims

1. In a solvent, hyaluronic acid or a salt thereof having a viscosity-average molecular weight greater than 3000 and not exceeding 500,000, hyaluronic acid having a viscosity-average molecular weight not exceeding 3000, glycerol, and a cryopreservation solution for hematopoietic cells, wherein the concentration of the hyaluronic acid having a viscosity-average molecular weight greater than 3000 and not exceeding 500,000 is 0.1 w / v% or more and 50 w / v% or less, the concentration of the hyaluronic acid having a viscosity-average molecular weight not exceeding 3000 is 0.1 w / v% or more and 10 w / v% or less, and the concentration of the glycerol is 1 w / v% or more and 60 w / v% or less, a cryopreservation solution for hematopoietic cells.

2. The cryopreservation solution for hematopoietic cells according to Claim 1, wherein the hyaluronic acid having a viscosity-average molecular weight greater than 3000 and not exceeding 500,000 has a viscosity-average molecular weight of 5000 or more.

3. The cryopreservation solution for hematopoietic cells according to Claim 1 or 2, wherein the hematopoietic cells are megakaryocyte progenitor cells.

4. The cryopreservation solution for hematopoietic cells according to Claim 1 or 2, wherein the hematopoietic cells are granulocytes, lymphocytes, monocytes, or erythrocytes.

5. The cryopreservation solution for hematopoietic cells according to Claim 1 or 2, wherein the hematopoietic cells are leukocytes.

6. A method for cryopreserving hematopoietic cells, characterized by suspending hematopoietic cells in the cryopreservation solution for hematopoietic cells according to any one of Claims 1 to 5 and cryopreserving them.

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