Cryopreservation solution

Abstract

To provide a cryopreservation solution for biological samples with a high cell survival rate, a cryopreservation method for biological samples, and a method for stably preserving biological samples over the long term.SOLUTION: The present invention provides a cryopreservation solution for biological samples that includes, in a solution, a macromolecule having a viscosity-average molecular weight of more than 3,000 and 500,000 or less and including a monomer with a hydrophilic group as a repeating unit or a salt of the macromolecule, and saccharides having a viscosity-average molecular weight of 3,000 or less or a salt of the saccharides, and a production method for the cryopreservation solution. The present invention also provides a cryopreservation method including a step for adding a cryopreservation solution to a biological sample, and a method for stably preserving biological samples over the long term.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a cryopreservation solution. The present invention also relates to a method for cryopreserving a biological sample using the cryopreservation solution, a method for preserving a biological sample, and a cryopreservation agent for a biological sample. [Background technology]

[0002] With the recent dramatic development of regenerative medicine research, regenerative medicine such as cell therapy is being actively conducted not only in humans but also in the veterinary field. Bone marrow-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells collected from living organisms are mass-produced after collection and used in the above-mentioned regenerative medicine and regenerative medicine research. In this case, it is common to freeze and store the surplus cells and use them as appropriate. There is also an increasing demand for a stable supply of such cells.

[0003] It is known that in the mechanism of cell cryopreservation, when ice crystals grow inside cells during the freezing and / or thawing process, the cell membrane and intracellular structures are damaged, and the cellular proteins are denatured, resulting in fatal damage to the cells. Therefore, when cryopreserving cells, it is important to prevent intracellular freezing. Usually, for cell cryopreservation, low molecular weight compounds such as dimethyl sulfoxide (DMSO), glycerin, and propylene glycol are used as intracellular permeable cryoprotective reagents by adding them to buffers such as culture media (Patent Document 1). Of these, DMSO is the most commonly used, and has a good effect on protecting cells and organelles. However, in intracellular permeable cryopreservation solutions, the low molecular weight compounds that are cryoprotective reagents penetrate into cells, raising concerns about the effect of the cryoprotective reagents on cells (Non-Patent Document 1).

[0004] Therefore, attempts have been made to use natural cryoprotectants as cryoprotective reagents instead of chemicals. For example, it is known that disaccharides, oligosaccharides, or high molecular weight polysaccharides are added to buffer solutions such as culture media as non-permeable cryoprotective reagents.

[0005] In addition, a method of retaining a biological component in a crosslinked body that forms a hydrogel is also being considered. Patent Document 2 describes a biological component preservative made from modified hyaluronic acid, in which a side chain having a substituent that reacts with a hydroxyl group to form a crosslinked structure is introduced into raw hyaluronic acid having a weight-average molecular weight of 5,000 to 4,000,000. The modified hyaluronic acid reacts with the hydroxyl groups of a compound having multiple hydroxyl groups, such as polyvinyl alcohol, to form a crosslinked product of the modified hyaluronic acid, and the biological component is embedded in this agar-like hydrogel to be used as a preservative. It is estimated that the molecular weight of the hydrogel described in Patent Document 2 as an actual preservative is several million or more. In Patent Document 2, the biological component is preserved in a refrigerator at about 4°C for a storage period of about several days.

[0006] Furthermore, Patent Document 3 describes a cryopreservation composition containing a carboxylated polyamino acid in which the amino groups of the polyamino acid are blocked by carboxylation (or acetylation) with a carboxylic acid anhydride, and an organic amphoteric agent. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Publication No. 63-216476 [Patent Document 2] International Publication No. 2016 / 076317 [Patent Document 3] Special Publication No. 2018-533377 [Non-patent literature]

[0008] [Non-Patent Document 1] REJUVENATION RESEARCH Volume 18 Number 5 (2015): 422-436, Mary Ann Liebert, Inc. Summary of the Invention [Problem to be solved by the invention]

[0009] Intracellular-permeating cryoprotectants promote the dehydration of cells, thereby slowing down the rate of intracellular ice crystal formation and inhibiting ice crystal formation. DMSO in particular is easy to permeate into cells, and is therefore effective for cryopreserving cells with complex structures such as mammalian cells, but as described in the above-mentioned Non-Patent Document 1, chemicals such as DMSO are cytotoxic. It is believed that as the penetration of cryoprotectants into cells progresses and the intracellular concentration increases, the toxic effects also increase.

[0010] Furthermore, it has been reported that DMSO induces differentiation of HL-60 cells and P19CL6 cells (derived from mouse embryonal carcinoma cells) (PNAS March 27, 2001 98 (7) 3826-3831. and Biochem Biophys Res Commun. 2004 Sep 24;322(3):759-65.), and also affects differentiation of ES cells (Cryobiology. 2006 Oct;53(2):194-205.). Therefore, the use of DMSO as a cryoprotectant is not suitable for cell preservation when it is necessary to maintain the undifferentiated state and functionality of stem cells. In addition, when storing samples for a long period of time using DMSO, it is essential to store the samples in liquid nitrogen or under an atmosphere that requires careful handling, which is considered to be an issue for the dissemination of regenerative medicine and regenerative medicine research.

[0011] On the other hand, natural cryoprotectants such as sugars have affinity for cells, but their molecular size is large and they are difficult to be taken up into cells. Therefore, it is thought that adding them from outside the cells alone is not enough to suppress intracellular freezing.

[0012] As mentioned above, the tested preservative for biological components described in Patent Document 2 has a storage period of only a few days, and it is considered that a better cryopreservation technique is required to store the components for several months. In addition, in order to produce the hydrogel described in Patent Document 2, it is necessary to introduce a modification group into hyaluronic acid, and further, it is necessary to use an additive to dissolve the gel in order to recover the biological components from the hydrogel after storage. However, if such an additive is introduced into the cryopreservation method, it can be used for research and testing, but it is difficult to use it for actual medical purposes due to the risk of impurities being mixed in.

[0013] Patent Document 3 discloses a cryopreservation composition containing a carboxylated polyamino acid in which the amino group of the polyamino acid is modified with a carboxylic acid, an organic amphoteric agent, and optionally a polysaccharide; however, the survival rate of cells cryopreserved was not sufficient.

[0014] Although a method using a non-permeable cryoprotective agent is also known, such a preservation agent alone cannot provide sufficient preservation effect, although it has little effect on cells. Therefore, products that combine intracellularly permeable compounds such as DMSO, glycerin, and propylene glycol with non-permeable substances to reduce or replace the amount of compounds are commercially available. However, no cryoprotective substance that shows good cryopreservation effect and is practical, using only natural components such as biological components, has yet to be reported.

[0015] The present invention has been made in view of the above problems, and provides a polymer aqueous solution for cryopreservation for appropriately preserving biological samples. In particular, the present invention aims to provide a polymer aqueous solution for cryopreservation that enables stable long-term preservation of biological samples at temperatures below -27°C without basically using chemical substances such as dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), etc., and without basically adding serum or serum-derived proteins, a cryopreservation agent for biological samples, and a method for stably freezing and preserving biological samples. [Means for solving the problem]

[0016] The present invention relates to a cryopreservation solution for biological samples, comprising a polymer or a salt thereof having a viscosity average molecular weight of more than 3000 and not more than 500,000, the polymer or salt 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 3000 in a solvent. The salt of the polymer or saccharide is preferably a metal salt, a halogen salt, or a sulfate salt. The metal salt is preferably a salt of an alkali metal or an alkaline earth metal. The alkali metal or alkaline earth metal may be selected from sodium, potassium, calcium, or the like. The halogen may be chlorine, bromine, or the like.

[0017] In the present invention, it is desirable that a polymer having a viscosity average molecular weight of more than 3000 and not more than 500,000, which contains a monomer having a hydrophilic group as a repeating unit, or a salt thereof, is contained as a main component, and a saccharide having a viscosity average molecular weight of not more than 3000 or a salt thereof is contained as a subcomponent. In this specification, the main component refers to the component having the highest weight ratio among the components dissolved in the solvent. Constituents other than the main component are subcomponents.

[0018] In the polymer used in the present invention, which contains a monomer having a hydrophilic group as a repeating unit, the hydrophilic groups are preferably not modified, or if modified, the number of the hydrophilic groups is 50% or less. It is presumed that the hydrophilic groups in the polymer are involved in protecting the biological sample, vitrifying the solvent, and replacing the sugar with a molecular weight of 3000 or less with the water around the biological sample, and if the hydrophilic groups are modified to become hydrophobic, the effect of cryopreserving cells is reduced. Therefore, it is preferable to exclude hydrophobic polymers such as carboxypolyamino acids as the main component.

[0019] The cryopreservation solution for biological samples of the present invention preferably does not contain dimethyl sulfoxide, which is an intracellularly penetrating cryoprotectant, and preferably does not contain cytotoxic cryoprotectants such as ethylene glycol, as these are harmful to cells after thawing.

[0020] 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.

[0021] A cryopreservation solution for a biological sample is preferably one in which the monomer having a hydrophilic group is a monomer having a hydrophilic group which is at least one selected from the group consisting of a hydroxyl group and a carboxylic acid group and salts thereof.

[0022] A cryopreservation solution for a biological sample 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 is preferred.

[0023] The cryopreservation solution for biological samples is preferably one in which the polymer is an alternating copolymer of the monomer having a hydrophilic group and the nitrogen-containing monomer.

[0024] A cryopreservation medium for a biological sample in which the monomer having a hydrophilic group is a monomer having a hydroxyl group substituted at an equatorial position is preferred.

[0025] A cryopreservation solution for biological samples containing a polymer having a viscosity average molecular weight of 10,000 or more or a salt thereof is preferred.

[0026] A cryopreservation medium for biological samples in which the polymer is a polymer containing multiple sugar residues is preferred.

[0027] A cryopreservation medium for biological samples in which the saccharide is a monosaccharide, a disaccharide, or an oligosaccharide is preferred.

[0028] A freezing storage medium for biological samples in which the sugar is glucose, fructose, galactose, or uronic acid in which the alcohol group of these sugars is oxidized, or an amino sugar in which the alcohol group is substituted with an amino group, sucrose, a cleavage product of a glycosaminoglycan, a constituent monosaccharide of a glycosaminoglycan, or a polymer or combination thereof is preferred.

[0029] A cryopreservation medium for biological samples in which the saccharide is glucuronic acid or N-acetylglucosamine is preferred.

[0030] A cryopreservation solution for biological samples in which the concentration of the polymer or a salt thereof is 5 w / v % or more and 20 w / v % or less is preferred.

[0031] A cryopreservation solution for biological samples in which the concentration of the sugar or its salt is 1 w / v % or more and 10 w / v % or less is preferred.

[0032] Cryopreservation solutions for biological samples that are non-intercellular-permeating cryopreservation solutions are preferred.

[0033] A cryopreservation medium for biological samples, wherein said biological sample is a cell, tissue, or tissue-like entity that is a membrane or aggregate, is preferred.

[0034] The freezing preservation medium is preferably for a biological sample, the biological sample being mesenchymal stem cells, blood cells, endothelial cells, or tissue for transplantation.

[0035] The freezing preservation medium is preferably for a biological sample, the biological sample being a sperm, an egg, or a fertilized egg.

[0036] The present invention also relates to a method for freezing a biological sample, which comprises cooling and freezing the biological sample using the cryopreservation solution of the present invention by a slow freezing method, desirably at a cooling rate of 10° C. / min or less, and preferably at a cooling rate of 1° C. / min or less.

[0037] The present invention also relates to a method for cryopreserving a biological sample, comprising the steps of: placing a biological sample in a cryopreservation solution containing, 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 3,000 or less; subjecting the cryopreservation solution containing the biological sample to freezing; and preserving the biological sample by maintaining the cryopreservation solution containing the biological sample at a temperature of -27°C or less.

[0038] The salt of the polymer or sugar is preferably a metal salt, a halogen salt, or a sulfate salt. As the metal salt, an alkali metal or an alkaline earth metal salt is preferably used. As the alkali metal or the alkaline earth metal, sodium, potassium, calcium, or the like can be selected. As the halogen, chlorine, bromine, or the like can be used. In addition, it is preferable that the cryopreservation solution does not contain a cell-permeable and cytotoxic compound such as dimethyl sulfoxide or ethylene glycol, because these are toxic to cells after thawing.

[0039] The viscosity average molecular weight of the polymer or its salt used in the method for cryopreservation of a biological sample of the present invention is desirably 400,000 or less, particularly 200,000 or less. This is because the viscosity can be adjusted to a low level and the solution is easy to handle as a cryopreservation solution.

[0040] In addition, the range of the freezing and preservation temperature in the method for freezing and preserving a biological sample of the present invention is not limited as long as it is −27° C. or lower, but the upper limit is desirably −70° C. or lower, preferably −80° C. or lower, and the lower limit is desirably −196° C. or higher, preferably −150° C. or higher.

[0041] A method for cryopreservation of a biological sample is preferred, wherein said protection of the biological sample occurs during storage.

[0042] In the method for cryopreserving a biological sample, the step of containing the biological sample in the cryopreservation solution is preferably carried out before cooling the biological sample.

[0043] The method for cryopreservation of a biological sample is preferred, wherein the biological sample is a cell, a tissue, or a tissue-like entity such as a membrane or an aggregate.

[0044] The method for freezing and preserving a biological sample is preferably such that the biological sample is a sperm, an egg, or a fertilized egg.

[0045] The present invention also relates to a method for preserving a biological sample, wherein the biological sample is preserved in the presence of a polymer or a salt thereof having a viscosity average molecular weight of more than 3,000 and not more than 500,000, the polymer 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 3,000 or less, and at a temperature of -27°C or lower, wherein the preservation is such that, when the biological sample is preserved in the presence of the polymer or the salt thereof and the saccharide or the salt thereof for a period of at least 5 months, the biological sample shows a decrease in viability of less than 5% based on the viability of the biological sample immediately before preservation.

[0046] The salt of the polymer or sugar is preferably a metal salt, a halogen salt, or a sulfate salt. As the metal salt, an alkali metal or an alkaline earth metal salt is preferably used. As the alkali metal or the alkaline earth metal, sodium, potassium, calcium, or the like can be selected. As the halogen, chlorine, bromine, or the like can be used. In addition, it is preferable that the cryopreservation solution does not contain a cell-permeable and cytotoxic compound such as dimethyl sulfoxide or ethylene glycol, because these are toxic to cells after thawing.

[0047] The viscosity average molecular weight of the polymer or its salt used in the method for preserving a biological sample of the present invention is desirably 400,000 or less, particularly 200,000 or less. This is because the viscosity can be adjusted to a low level and the solution is easy to handle as a cryopreservation solution.

[0048] In addition, the range of the freezing storage temperature in the method for preserving a biological sample of the present invention is not limited as long as it is −27° C. or lower, but the upper limit is desirably −70° C. or lower, preferably −80° C. or lower. The lower limit is desirably −196° C. or higher, preferably −150° C. or higher.

[0049] A preferred method for preserving a biological sample is characterized in that the biological sample shows a decrease in viability of less than 10% based on the viability of the biological sample immediately before storage when stored for a period of at least 6 months in the presence of the polymer or its salt and the saccharide or its salt.

[0050] A preferred method for preserving a biological sample is characterized in that when a biological sample is thawed after frozen storage at -27°C or below and then stored at 4°C for one day, the biological sample shows a decrease in viability of less than 5% based on the viability of the biological sample immediately after thawing.

[0051] The method for preserving a biological sample, wherein the biological sample is a cell, is preferred.

[0052] A method for preserving a biological sample, wherein said biological sample is a mammalian cell, is preferred.

[0053] A method for preserving a biological sample is preferred, wherein said biological sample is a mammalian mesenchymal stem cell, a mammalian blood cell, or a mammalian endothelial cell.

[0054] The present invention also relates to use of a mixture of a polymer having a viscosity average molecular weight of more than 3000 and not more than 500,000, which contains a monomer having a hydrophilic group as a repeating unit, or a salt thereof, and a saccharide having a viscosity average molecular weight of 3000 or less, or a salt thereof, for preserving a biological sample, preferably in vitro, at a temperature of not more than -27°C, preferably more than -150°C and not more than -70°C, preferably more than -150°C and not more than -80°C.

[0055] The salt of the polymer or sugar is preferably a metal salt, a halogen salt, or a sulfate salt. As the metal salt, an alkali metal or an alkaline earth metal salt is preferably used. As the alkali metal or the alkaline earth metal, sodium, potassium, calcium, or the like can be selected. As the halogen, chlorine, bromine, or the like can be used. In addition, it is preferable that the cryopreservation solution does not contain a cell-permeable and cytotoxic compound such as dimethyl sulfoxide or ethylene glycol, because these are toxic to cells after thawing.

[0056] The present invention relates to use of a mixture of a polymer or a salt thereof having a viscosity average molecular weight of more than 3,000 and not more than 500,000, the polymer 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 3,000 or less, for cryopreservation of a biological sample.

[0057] The salt of the polymer or sugar is preferably a metal salt, a halogen salt, or a sulfate salt. As the metal salt, an alkali metal or an alkaline earth metal salt is preferable. As the alkali metal or the alkaline earth metal, sodium, potassium, calcium, etc. can be selected. As the halogen, chlorine, bromine, etc. can be used.

[0058] The viscosity average molecular weight of the polymer or its salt used in the present invention is desirably 400,000 or less, particularly 200,000 or less, because the viscosity can be adjusted to a low level and the polymer or salt can be easily handled as a cryopreservation solution.

[0059] The range of the cryopreservation temperature is not limited as long as it is −27° C. or lower, but the upper limit is desirably −70° C. or lower, preferably −80° C. or lower. The lower limit is desirably −196° C. or higher, preferably −150° C. or higher.

[0060] The present invention also relates to a cryopreservation agent for biological samples, comprising a mixture of a polymer or a salt thereof having a viscosity average molecular weight of more than 3,000 and not more than 500,000, the polymer 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 3,000 or less.

[0061] The salt of the polymer or sugar is preferably a metal salt, a halogen salt, or a sulfate salt. As the metal salt, an alkali metal or an alkaline earth metal salt is preferable. As the alkali metal or the alkaline earth metal, sodium, potassium, calcium, etc. can be selected. As the halogen, chlorine, bromine, etc. can be used.

[0062] The viscosity average molecular weight of the polymer or its salt used in the present invention is desirably 400,000 or less, particularly 200,000 or less, because the viscosity can be adjusted to a low level and the polymer or salt can be easily handled as a cryopreservation solution.

[0063] The range of the cryopreservation temperature is not limited as long as it is −27° C. or lower, but the upper limit is desirably −70° C. or lower, preferably −80° C. or lower. The lower limit is desirably −196° C. or higher, preferably −150° C. or higher.

[0064] Preferably the polymer comprises a pentose, hexose or uronic acid or combinations thereof as repeating units.

[0065] It is preferred that the polymer further comprises an amino sugar as a repeat unit.

[0066] The hydrophilic group of the polymer or its salt is preferably not modified.

[0067] Preferably, the polymer is hyaluronic acid, dextran, pullulan, or chondroitin sulfate. As polymers, carboxylated polyamino acids are preferably excluded.

[0068] Preferably, the saccharide is a monosaccharide, disaccharide, or oligosaccharide.

[0069] The sugar is preferably glucose, fructose, galactose, or uronic acid in which the alcohol group of these sugars is oxidized, or an amino sugar in which the alcohol group is substituted with an amino group, sucrose, a cleavage product of a glycosaminoglycan, a constituent monosaccharide of a glycosaminoglycan, or a polymer or combination thereof.

[0070] The present invention also relates to a method for producing a cryopreservation solution for biological samples, which comprises a polymer or a salt thereof having a viscosity average molecular weight of more than 3,000 and not more than 500,000, the polymer 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 3,000 or less, the method comprising dissolving the polysaccharide or its salt having a molecular weight of more than 500,000 in water, and then performing an extraction treatment under subcritical conditions of water.

[0071] The salts of the polymers and / or saccharides used in the present invention are preferably metal salts, halogen salts, or sulfate salts. The metal salts are preferably salts of alkali metals or alkaline earth metals. The alkali metals or alkaline earth metals may be selected from sodium, potassium, calcium, etc. The halogens may be chlorine, bromine, etc.

[0072] The viscosity average molecular weight of the polymer or its salt used in the present invention is desirably 400,000 or less, particularly 200,000 or less, because the viscosity can be adjusted to a low level and the polymer or salt can be easily handled as a cryopreservation solution.

[0073] The method for producing a cryopreservation solution for biological samples is preferably one in which the subcritical conditions for water are a temperature of 150 to 350° C. and a pressure of 0.5 to 25 MPa.

[0074] According to the present invention, biological samples such as cells and tissues can be appropriately preserved without using cell-permeable and toxic compounds such as DMSO and ethylene glycol, which are intracellular-permeable cryoprotectants. Furthermore, since the cryopreservation solution of the present invention is intracellularly non-permeable, it has low toxicity to cells despite showing a high cryopreservation effect.

[0075] In addition, because it does not contain serum or serum-derived proteins, it is not contaminated by bacteria or viruses. However, it is possible to add proteins that are not contaminated by bacteria or viruses. It is also possible to add chemical substances that are cytotoxic at low concentrations that do not impair the function of cells.

[0076] The "viscosity average molecular weight" of the polymer or saccharide used in the present invention is determined by the following method and formula.

[0077] Intrinsic viscosity measurement: (1) Dissolve a specified amount of NaCl in ion-exchanged water at 30°C to prepare a 0.2 M NaCl solution (standard solution). (2) Dissolve a polymer or sugar sample in a standard solution at 30°C to prepare a stock solution. Measure the viscosity of each of the standard solution and the stock solution, and adjust the relative viscosity of the stock solution to 2.0 to 2.4 with respect to the standard solution. (3) Dilute the 30°C stock solution 5 / 4, 5 / 3, and 5 / 2 times with the 30°C standard solution. (4) Measure the viscosity of the standard solution, undiluted solution, and diluted solution at 30°C. Use an E-type viscometer to measure the viscosity. (5) The viscosity of the original solution and the diluted solution divided by the viscosity of the standard solution is the relative viscosity (η r ) and derive the reduced viscosity based on the following formula: TIFF2024178397000001.tif2146 where η sp : Reduced viscosity of polymer or sugar [mL / g], η r : relative viscosity of polymer or sugar [-], C: concentration of polymer or sugar [g / mL]. (6) Plot the relationship between the concentration of the polymer or sugar and the reduced viscosity of the polymer or sugar, and draw an approximate straight line. The intercept of the approximate straight line (where the concentration of the polymer or sugar = 0) is the limiting viscosity.

[0078] Viscosity average molecular weight: The viscosity average molecular weight is calculated from the intrinsic viscosity. TIFF2024178397000002.tif3559The viscosity average molecular weight M is calculated from the above Mark Hoying-Sakurada equation using the intrinsic viscosity derived from measurement and the values ​​of K and α published in literature, etc.

[0079] K and α are values ​​that vary depending on the type of polymer. The values ​​of K and α are disclosed in many published documents, such as the "Polymer Materials Handbook" (compiled by the Society of Polymer Science). The viscosity average molecular weight can be calculated using these published values.

[0080] 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, and K = 5.8 × 10 for chondroitin sulfate. -4 , α=0.74 can be used.

[0081] In the case of monosaccharides, disaccharides, and compounds that are considered to be monomolecular, the molecular weight is clearly specified from the structural formula, and therefore in the present invention, the molecular weight specified from the structural formula is treated as a fictitious viscosity average molecular weight.

[0082] In addition, as the solvent used to prepare the cryopreservation solution of the present invention, it is preferable to use an aqueous solvent such as water. In particular, it is preferable to use an isotonic solution in which the salt concentration and sugar concentration are adjusted by sodium ions, potassium ions, calcium ions, etc. so that the osmotic pressure is approximately the same as that of body fluids or cell fluids. Specifically, for example, water, physiological saline, physiological saline with a buffer effect such as phosphate buffered saline (PBS), Dulbecco's phosphate buffered saline, Tris buffered saline (TBS), HEPES buffered saline, etc., balanced salt solutions such as Hanks' balanced salt solution, Ringer's solution, lactate Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution, or animal cell culture basal media 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. Effect of the Invention

[0083] The aqueous polymer solution for cryopreservation of biological samples of the present invention uses a polymer component with a viscosity-average molecular weight of more than 3000 and not more than 500,000 to prevent ice crystals from forming in the solvent portion and vitrify the solution, thereby suppressing the rupture of biological samples such as cells due to ice crystal formation, and by replacing water molecules near the boundary tissue of biological samples such as cell membranes with low molecular weight sugars with a viscosity-average molecular weight of not more than 3000, the formation and growth of ice crystals near the cell membrane can be inhibited, and cell membrane damage can be suppressed. Furthermore, although the cryopreservation solution for biological samples of the present invention is an intracellular non-permeable cryopreservation solution, it exhibits a high cryopreservation effect that could not be obtained by conventional techniques, and therefore damage to the biological sample and changes in the properties of the biological sample can be significantly reduced.

[0084] In addition, the polymer aqueous solution for cryopreservation of biological samples of the present invention does not contain intracellularly penetrating chemicals such as DMSO and ethylene glycol, so that it is possible to reduce cytotoxicity and the effects on cell properties as reported for DMSO. Therefore, the properties of the biological sample can be maintained during and after cryopreservation. It is possible to add cytotoxic chemicals such as DMSO and ethylene glycol at low concentrations that do not impair the function of cells.

[0085] 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.

[0086] In addition, because it does not contain serum and / or serum-derived proteins, it is not subject to bacterial or viral contamination, although it is possible to add proteins that are not contaminated by bacteria or viruses.

[0087] In addition, since the polymer aqueous solution for cryopreservation of biological samples of the present invention has a glass transition point at about -23°C±4°C, according to the method for cryopreservation of biological samples and the method for preserving biological samples of the present invention, the frozen biological sample can be stably preserved for a long period of time at a temperature of -27°C or lower, i.e., in a deep freezer, without using cytotoxic chemicals such as DMSO or ethylene glycol or proteins such as serum. It is possible to add proteins that are not contaminated by bacteria or viruses. It is also possible to add cytotoxic chemicals such as DMSO or ethylene glycol at low concentrations that do not impair the function of cells.

[0088] The range of the cryopreservation temperature is not limited as long as it is −27° C. or lower, but the upper limit is desirably −70° C. or lower, preferably −80° C. or lower. The lower limit is desirably −196° C. or higher, preferably −150° C. or higher. [Brief description of the drawings]

[0089] [Figure 1] FIG. 1 is a graph showing the cell viability of primary human mesenchymal stem cells cryopreserved using the test cryopreservation solution of the present invention. [Figure 2A] FIG. 1 shows the results of analysis of a frozen storage solution by differential scanning calorimetry. [Figure 2B] FIG. 2 is an enlarged view showing the analysis results of a cryopreservation solution by differential scanning calorimetry. [Figure 3A] FIG. 1 shows the results of differential scanning calorimetry analysis of a frozen storage solution containing ultrapure water. [Figure 3B] FIG. 1 shows the results of differential scanning calorimetry analysis of a cryopreservation solution containing DMSO as a cryoprotectant. [Figure 3C] FIG. 2 is a diagram showing the analysis results of a frozen storage solution containing a test sample of Example 1 by differential scanning calorimetry. [Figure 4A] FIG. 1 shows the long-term preservation effect of primary human mesenchymal stem cells cryopreserved using the test cryopreservation solution of the present invention. [Figure 4B] FIG. 1 shows the long-term preservation effect of primary human mesenchymal stem cells during refrigerated preservation using the test cryopreservation solution of the present invention. [Figure 5A] FIG. 1 shows the amount of HGF produced in primary human mesenchymal stem cells after cryopreservation using the test cryopreservation solution of the present invention. [Figure 5B] FIG. 1 shows the amount of IL-10 produced in primary human mesenchymal stem cells after cryopreservation using the test cryopreservation solution of the present invention. [Figure 6] FIG. 1 shows the expression levels of undifferentiation biomarkers in primary human mesenchymal stem cells after cryopreservation using the test cryopreservation solution of the present invention. [Figure 7] FIG. 1 shows the preservation effect of cryopreservation solutions containing various polymers of the present invention on primary canine mesenchymal stem cells. [Figure 8] FIG. 1 shows the cell protective effect of the saccharides of the present invention. [Figure 9A] FIG. 1 shows the intracellular vitrification state after freezing in a control experiment. [Figure 9B]FIG. 2 shows the intracellular vitrification state after freezing using a test cryopreservation solution of Comparative Example 1. [Figure 9C] FIG. 2 shows the state of intracellular vitrification using the test cryopreservation solution of Example 1. [Figure 10A] FIG. 13 is a diagram showing the brightness difference in the intracellular vitrification state. [Figure 10B] The brightness difference within the cell in the vitrified state was converted into a numerical value according to the Munsell scale (0 to 10), and this is a graph showing the brightness difference between the solvent and the inside of the cell. [Figure 11] FIG. 1 shows the cell area of ​​frozen primary canine mesenchymal stem cells in a cryopreservation solution. [Figure 12] FIG. 1 shows the cryoprotective effect on various cells during cryopreservation using the test cryopreservation solution of the present invention. [Figure 13] FIG. 1 is a graph showing the cell protective effect of a combination of a polymer and a saccharide according to the present invention. [Figure 14] FIG. 1 is a graph showing the cell protective effect of a combination of a polymer and a saccharide according to the present invention. [Figure 15] FIG. 1 is a graph showing the cell protective effect of a combination of a polymer and a saccharide according to the present invention. [Figure 16] FIG. 1 shows the cell viability of primary canine mesenchymal stem cells cryopreserved using the saccharides of the present invention. [Figure 17A] FIG. 1 shows the results of HPLC analysis of test samples from various production examples. [Figure 17B] FIG. 1 shows the results of HPLC analysis of test samples from various production examples. [Figure 17C] FIG. 1 shows the results of HPLC analysis of test samples from various production examples. [Figure 17D] FIG. 1 shows HPLC analysis results for a calibration curve of saccharides having a given number of sugars. [Figure 18] FIG. 2 shows the results of HPLC analysis of a test sample having a polymer chain of the present invention. [Figure 19] FIG. 1 shows cell viability during cryopreservation using gelatin as a test sample. [Figure 20]FIG. 1 is a graph showing cell viability when carboxylated polylysine and carboxylated polylysine to which hyaluronic acid having a viscosity average molecular weight of 1,000 has been added were used as cryopreservation agents in a cell cryopreservation solution. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0090] The aqueous solution of a polymer or its salt for cryopreservation of a biological sample of the present invention (hereinafter also referred to as the aqueous polymer solution of the present invention) is a cryopreservation solution containing a polymer or its salt having a viscosity-average molecular weight of more than 3000 and not more than 500,000, and a saccharide or its salt having a viscosity-average molecular weight of 3000 or less. The polymer of the present invention is a polymer containing a monomer having a hydrophilic group as a repeating unit. That is, the cryopreservation solution of a biological sample of the present invention is a solution in which a cryopreservation agent containing a monomer having a hydrophilic group as a repeating unit and a mixture of a polymer or its salt having a viscosity-average molecular weight of more than 3000 and not more than 500,000, and a saccharide or its salt having a viscosity-average molecular weight of 3000 or less is dissolved in a solvent.

[0091] The viscosity average molecular weight of the polymer or its salt used in the present invention is desirably 400,000 or less, particularly 200,000 or less, because the viscosity can be adjusted to a low level and the polymer or salt can be easily handled as a cryopreservation solution.

[0092] The "viscosity average molecular weight" of the polymer or saccharide used in the present invention means a value calculated by the following method and formula.

[0093] The method for measuring the intrinsic viscosity and the method for calculating the viscosity average molecular weight using the intrinsic viscosity are described below. Intrinsic viscosity measurement: (1) Dissolve a specified amount of NaCl in ion-exchanged water at 30°C to prepare a 0.2 M NaCl solution (standard solution). (2) Prepare a stock solution by dissolving the polymer or sugar sample in a standard solution at 30°C. If the polymer or sugar sample is obtained in the form of a solution, the solid portion obtained by removing the solvent from the solution is used as the polymer or sugar sample. In the case of a mixed sample of polymers and sugars, a mixed sample containing multiple polymers, or a mixed sample containing multiple sugars, each substance is separated and fractionated, and the solvent is removed from each substance to obtain the polymer or sugar sample. If the polymer and / or sugar are unknown, the substance is identified for the polymer and / or sugar by HPLC, LC-MS, LC-IR, etc. If multiple unknown polymers and / or sugars are included, each component is separated and fractionated, and the substance for each polymer and / or sugar is identified by HPLC, LC-MS, LC-IR, etc., and the viscosity average molecular weight is calculated as described below. If the mixture contains impurities in the polymer or sugar, but the viscosity is not affected (for example, impurities such as metal salts), the mixture is used as the polymer or sugar sample. If the solution contains impurities that affect the calculation of the viscosity average molecular weight, remove the impurities or separate the polymers and sugars before measuring. Measure the viscosity of the standard solution and the stock solution, and adjust the relative viscosity of the stock solution to 2.0 to 2.4 compared to the standard solution. (3) Dilute the 30°C stock solution 5 / 4, 5 / 3, and 5 / 2 times with the 30°C standard solution. (4) Measure the viscosity of the standard solution, undiluted solution, and diluted solution at 30°C. Use an E-type viscometer to measure the viscosity. (5) The viscosity of the original solution and the diluted solution divided by the viscosity of the standard solution is the relative viscosity (η r ) and derive the reduced viscosity based on the following formula: TIFF2024178397000003.tif2146Here, η sp : Reduced viscosity of polymer or sugar [mL / g], η r : relative viscosity of polymer or sugar [-], C: concentration of polymer or sugar [g / mL]. (6) Plot the relationship between the concentration of the polymer or sugar and the reduced viscosity of the polymer or sugar, and draw an approximate straight line. The intercept of the approximate straight line (where the concentration of the polymer or sugar = 0) is the limiting viscosity. Viscosity average molecular weight: The viscosity average molecular weight is calculated from the intrinsic viscosity. TIFF2024178397000004.tif3559The viscosity average molecular weight M is calculated from the intrinsic viscosity obtained by measurement and the values ​​of K and α published in the literature, etc., in the Mark Hoying-Sakurada formula above. In the case of hyaluronic acid, K = 3.6 × 10 -4 and α = 0.78 to calculate the viscosity average molecular weight M. For 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 K = 5.8 × 10 for chondroitin sulfate. -4 , α = 0.74, and for carboxypolylysine, K = 2.78 × 10 -5 , α=0.87 is used.

[0094] K and α are values ​​that vary depending on the type of polymer. The values ​​of K and α are disclosed in many published documents, such as the "Polymer Materials Handbook" (compiled by the Society of Polymer Science). The viscosity average molecular weight is calculated using these published values.

[0095] In the present invention, the molecular weight calculated by this method is regarded as the viscosity average molecular weight. In the case of monosaccharides and disaccharides such as sucrose and glucuronic acid, and compounds considered to be monomolecular, the molecular weight is clearly specified from the structural formula, so the molecular weight specified from the structural formula is treated as the viscosity average molecular weight.

[0096] The cryopreservation solution of the present invention is a preservation solution for appropriately preserving a biological sample when the biological sample is cryopreserved. By containing a polymer having a predetermined molecular weight and a large number of hydrophilic groups, the polymer aqueous solution of the present invention can trap solvent molecules in a matrix formed by the polymer chain during the cooling process. Since the polymer chain contains hydrophilic groups, the action of the polymer chain restricts the molecular motion of the solvent water during cooling, and the water can be solidified and / or frozen in a vitrified state without crystallizing. Usually, a freezing method called vitrification is a method in which, when a solution freezes, a solute (a cryoprotectant for preventing freezing damage) is excluded from the crystals, increasing the salt concentration in the remaining solution, and an osmotic pressure difference occurs between the inside and outside of the cell, dehydrating the inside of the cell and vitrifying the inside of the cell, and is applied to cells with a particularly low survival rate after thawing. In such a method, in order to make water more easily vitrified, the concentration of the solute (cryoprotectant) is increased or the cooling rate is increased. However, it has been known that increasing the osmotic pressure difference increases damage to cells, and that recrystallization occurs during thawing, causing damage to the cells. In addition, the method is difficult to perform.

[0097] According to the cryopreservation solution containing the polymer of the present invention, the inside of the cells is dehydrated and vitrified by the action of the polymer chain, so that the formation of ice crystals in the cells is suppressed, and furthermore, the osmotic shock in the cells during freezing, which is a problem in the conventional vitrification method, can be weakened. Therefore, it is not necessary to contain chemical substances such as dimethyl sulfoxide (DMSO) or ethylene glycol, which have cytotoxicity, and the cryopreservation solution containing the polymer of the present invention does not contain DMSO and / or ethylene glycol. When the cryopreservation solution of the present invention is cooled, the amorphous glass state in the frozen state is stabilized by the viscosity average molecular weight of the polymer of the present invention being more than 3000 and not more than 500000. Therefore, the cells are less likely to be damaged by cooling and freezing, and the cells can be cryopreserved stably and efficiently. Therefore, the survival rate of the cells in the biological sample 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. Furthermore, if the viscosity average molecular weight of the polymer is greater than 500,000, the viscosity increases significantly, the solubility decreases, and the prepared solution foams, which may cause problems such as poor handling. The viscosity average molecular weight is preferably, for example, 10,000 or more. Furthermore, a polymer having a viscosity average molecular weight of 400,000 or less, or even 200,000 or less, is preferable, and 150,000 or less is particularly preferable. This is because the viscosity can be adjusted low and it is easy to handle as a frozen storage solution.

[0098] The polymer used in the present invention is a polymer containing a monomer having a hydrophilic group as a repeating unit. The hydrophilic group is, for example, a hydroxyl group and a carboxylic acid group and a salt thereof. The polymer of the present invention may also contain a nitrogen-containing monomer having an optionally substituted amino group or an optionally substituted amide group as a repeating unit. Furthermore, the polymer of the present invention preferably has a hydroxyl group at an equatorial position in its structure. This is believed to allow the water of the solvent to be more effectively trapped in the matrix formed by the polymer chains during freezing.

[0099] The monomer having a hydrophilic group is, for example, a sugar residue. In this case, the polymer of the present invention may be a polymer containing a repeating unit of sugar residues bound 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 are substituted, for example, monosaccharides in which the hydroxyl and / or hydroxymethyl groups are substituted with at least one substituent selected from the group consisting of a carboxyl group, an amino group, an N-acetylamino group, a sulfoxy group, a methoxycarbonyl group, and a carboxymethyl group.

[0100] Examples of monosaccharides include triose, tetrose, pentose, hexose, and heptose. For example, examples of pentoses include ribose, arabinose, xylose, lyxose, xylulose, ribulose, and deoxyribose. Examples of hexoses include glucose, mannose, galactose, fructose, sorbose, tagatose, fucose, fuculose, and rhamnose.

[0101] For example, the monosaccharide substituted with a carboxyl group includes uronic acid. Examples of the uronic acid include glucuronic acid, iduronic acid, mannuronic acid, and galacturonic acid. Examples of the monosaccharide substituted with an amino group include amino sugars. Examples of the amino sugar include glucosamine, galactosamine, mannosamine, and muramic acid. Examples of the monosaccharide substituted with an N-acetylamino group include N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, and N-acetylmuramic acid. Examples of the monosaccharide substituted with a sulfooxy group include galactose-3-sulfate. Examples of the monosaccharide having multiple substituents include N-acetylglucosamine-4-sulfate, iduronic acid-2-sulfate, glucuronic acid-2-sulfate, N-acetylgalactosamine-4-sulfate, neuraminic acid, and N-acetylneuraminic acid.

[0102] For example, the polymer used in the present invention is a polymer that contains the above-mentioned monosaccharide as a repeating unit. For example, the polymer of the present invention may be a polymer that contains pentose, hexose, or uronic acid, or a combination thereof, which may be substituted, as a repeating unit. 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. It may also be a sulfated polysaccharide in which one or more hydroxyl groups are substituted with sulfooxy groups. Although not limited thereto, examples of the polymer of the present invention include hyaluronic acid, dextran, pullulan, and chondroitin sulfate.

[0103] The polymer used in the present invention may be naturally derived or may be chemically synthesized. Commercially available polymers may be used as they are. A naturally derived polymeric compound or a commercially available polymeric compound having a larger molecular weight may be used, and the cleavage product may be obtained by subjecting the compound to hydrolysis, enzyme treatment, subcritical treatment, or other treatment, and the molecular weight may be adjusted to obtain the polymer of the present invention. Each monomer may also be naturally derived, may be a naturally derived monomer that has been modified or substituted, or may be chemically synthesized. For example, preferably, the monomer contained in the polymer of the present invention is a biological component. It is considered that the cytotoxicity of a cryopreservation solution containing the polymer is low.

[0104] The hydrophilic groups of the polymer used in the present invention are preferably not modified, or modified but not exceeding 50% of the total number of hydrophilic groups, i.e., no substituents are introduced into the polymer chain, or even if they are introduced, the number is preferably not exceeding 50% of the total number of hydrophilic groups. It is presumed that the hydrophilic groups of the polymer, particularly the OH group, NH2 group, and COOH group, contribute to the protection of the biological sample, the vitrification of the solvent, and the replacement of sugar with water around the cells, and therefore it is advantageous to improve the survival rate of the biological sample if these functional groups are not modified. Furthermore, there is a risk that the reagents used to modify the hydrophilic groups of the polymer may adversely affect the cryoprotective effect of the polymer or may make it impossible to use the polymer for cryopreservation of biological samples for regenerative medicine purposes.

[0105] In addition, it is believed that the hydrophilic groups of polymers can hold low molecular weight sugars through hydrogen bonds, and it is presumed that the presence of polymers holding low molecular weight sugars around biological samples such as cells can promote the replacement of water molecules and sugars near the cell membrane. Therefore, if the hydrophilic groups are modified, the retention effect of the hydrophilic groups on low molecular weight sugars is reduced, and even if low molecular weight sugars coexist, they do not contribute sufficiently to improving the survival rate of biological samples. For example, it is not preferable to modify OH groups or NH2 groups with carboxylic acids, etc.

[0106] The salt of the polymer or saccharide used in the present invention may be a metal salt, a halogen salt or a sulfate salt of the polysaccharide. As the metal salt, an alkali metal or an alkaline earth metal salt is preferable. As the alkali metal or the alkaline earth metal, sodium, potassium, calcium, etc. are selected. As the halogen, chlorine, bromine, etc. can be used. It is considered that the salt lowers the freezing point of the solvent, thereby contributing to the vitrification of the solvent.

[0107] The cryopreservation solution and cryopreservation agent of the present invention contain a saccharide or its salt having a viscosity average molecular weight of 3000 or less. When the cryopreservation solution and cryopreservation agent contain a saccharide having such a small molecular weight together with a polymer, the water molecules near the cell membrane are replaced by the saccharide, and the formation and growth of ice crystals near the cell membrane are inhibited, and as a result, cell membrane damage can be significantly inhibited. 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 with a molecular weight of 3000 or less, preferably 2000 or less, more preferably 1000 or less.

[0108] The saccharide may be, for example, the monosaccharide described above as a monomer constituting a polymer. For example, the saccharide may be glucose, fructose, galactose, or uronic acid in which the alcohol group of these is oxidized, or an amino sugar in which the alcohol group is substituted with an amino group, sucrose, trehalose, or a polymer or combination thereof. The saccharide may also be, for example, a fragment of the polymer used in the present invention, such as hyaluronic acid, dextran, pullulan, or chondroitin sulfate. Although not particularly limited as long as it does not impair the effect of the present invention, the saccharide may be, for example, a cleavage product (fragment) of glycosaminoglycan, that is, a monosaccharide constituting glycosaminoglycan, a disaccharide thereof, or an oligosaccharide thereof.

[0109] Preferably, saccharide is the cleavage product of hyaluronic acid.Therefore, preferably, saccharide used in the present invention is glucuronic acid or N-acetylglucosamine, or the disaccharide or oligosaccharide thereof.Preferably, saccharide can be glucuronic acid or its modified compound, or the disaccharide or oligosaccharide thereof.

[0110] The term "cleavage product" as used in the present invention means a compound having a smaller molecular weight than the original polymer, which is considered to be obtained when a polymer is subjected to hydrolysis, enzyme treatment, subcritical treatment, or the like. The polymer used in the present invention may be a polymer having a viscosity average molecular weight of more than 3000 and not more than 500,000 obtained by treating a larger polymer compound, as described above, and the saccharide used in the present invention may be a saccharide having a viscosity average molecular weight of not more than 3000 obtained by treating a polymer. The viscosity average molecular weight of the polymer or its salt used in the present invention is preferably not more than 400,000, particularly not more than 200,000. This is because the viscosity can be adjusted to a low level and it is easy to handle as a frozen storage solution. The cleavage product may be a monomer that is a component of the original polymer and / or a polymer of various polymerization degrees of the monomer and / or a mixture thereof.

[0111] "Subcritical processing" means contacting the raw material to be extracted with a subcritical fluid as an extracting solvent that has been brought into a subcritical state under conditions of a predetermined temperature and a predetermined pressure. For example, when water is raised to a pressure of 22.12 MPa or more and a temperature of 374.15°C or more, it is in a state that is neither liquid nor gas. This point is called the critical point of water, and hot water with a temperature and pressure close to the critical point is called subcritical water. Using the hydrolysis action of this subcritical water, desired components can be obtained from the raw material to be extracted. Conditions for subcritical processing in the present invention include, for example, a temperature of 150°C or more and 350°C or less, and the subcritical processing pressure can be equal to or higher than the saturated vapor pressure at each temperature, but can be, for example, 0.5 MPa or more and 25 MPa or less. After the subcritical processing, components with a molecular weight of less than a predetermined value are separated and collected, and can be used as the cleavage product in the present invention. In addition, there are no particular limitations on the hydrolysis or enzyme processing, and reagents and processing methods that are commonly used can be used without problems.

[0112] The polymer and saccharides having a predetermined molecular weight in the present invention may be obtained simultaneously by a single subcritical treatment. That is, the polymer and saccharides in the present invention may be a subcritically treated product of a polymer compound having a first molecular weight distribution in a molecular weight range of more than 3000 and not more than 500000 as a viscosity average molecular weight, and a second molecular weight distribution in a molecular weight range of not more than 3000 as a viscosity average molecular weight. Therefore, for example, the method for producing a cryopreservation solution for a biological sample of the present invention may include a step of obtaining the polymer and saccharides of the present invention by dissolving a polymer, which is a polysaccharide having a molecular weight of more than 500000 as a viscosity average molecular weight, in water and then performing an extraction treatment under subcritical conditions of water. However, the polymer and saccharides of the present invention may be used in combination with those treated and / or separated based on molecular weight in separate treatment steps. The viscosity average molecular weight of the polymer or its salt contained in the cryopreservation solution of the present invention is preferably 400000 or less, particularly 200000 or less. This is because the viscosity can be adjusted to be low, making it easy to handle as a cryopreservation solution.

[0113] The cryopreservation solution containing the polymer and sugars of the present invention has low cytotoxicity, and furthermore, since there is no need to increase the concentration of solutes to restrict the molecular motion of water, it is considered that damage to cells is low. Moreover, unlike conventional vitrification methods, the cryopreservation solution of the present invention does not require a high cooling rate during cooling to reduce osmotic shock. Therefore, the cryopreservation solution of the present invention can efficiently freeze and preserve biological samples in a simple method with reduced toxicity.

[0114] According to the results of thermal analysis of the cryopreservation solution of the present invention by differential scanning calorimetry (DSC), the cryopreservation solution of the present invention has a glass transition temperature in the vicinity of -23°C ± 4°C. Furthermore, the cryopreservation solution of the present invention not only suppresses ice crystal formation during the cooling process, but also suppresses recrystallization of water during the subsequent heating process, i.e., during melting. That is, in cryopreservation using the polymer and saccharide of the present invention, the glassy state of the frozen biological sample is believed to be stabilized. Since the glassy state is further stabilized in this way, the cryopreservation solution of the present invention exhibits a high cell protection effect without the need for the addition of protein components such as serum derived from human or bovine or serum-derived components (e.g., albumin, etc.). Therefore, there is no need to worry about infections, and it is believed that it is not affected by lot-to-lot differences due to biological preparations. Since the cytotoxicity is also low, thawed cells after cryopreservation using the cryopreservation solution of the present invention exhibit a high survival rate. It is possible to add proteins that do not pose a risk of infection.

[0115] As described above, the cryopreservation method using the cryopreservation solution of the present invention does not require a large cooling rate for cell protection during cooling, so after adding the cryopreservation solution of the present invention to a biological sample, the sample is cooled to -27 ° C or lower, which is the glass transition temperature of the cryopreservation solution of the present invention. For example, the cryopreservation solution of the present invention containing a biological sample is placed in a freezing treatment container or the like and left in a deep freezer at -80 ° C, and cells, tissues, etc. to be preserved can be stably frozen and preserved. Since a low temperature such as -150 ° C, which is required for cryopreservation of normal cells, is not necessarily required, it is possible to eliminate the need for special containers for liquid nitrogen cryopreservation and the preparation of liquid nitrogen. Therefore, the operation related to the cryopreservation of biological samples can be significantly simplified. In addition, the cryopreservation solution of the present invention can also suppress recrystallization of water during thawing, so if the cryopreservation solution of the present invention is used, a series of steps of freezing, preserving, and thawing a biological sample can be easily and efficiently performed without the need for special procedures. Of course, it is also possible to freeze and preserve a biological sample using the cryopreservation solution of the present invention and liquid nitrogen.

[0116] Furthermore, the cryopreservation solution of the present invention is a non-permeable cryoprotective reagent, so it is considered to have low cytotoxicity. In addition, the cryopreservation solution of the present invention is considered to have low cytotoxicity, so that the sugars can function for cell protection, so that the properties of cells are not changed during cryopreservation. Therefore, it is considered that biological samples can be cryopreserved while maintaining the properties of cells.

[0117] The cryopreservation solution of the present invention contains the polymer or its salt at a concentration of about 1 w / v% or more and 50 w / v% or less. If the concentration is lower than 5 w / v%, the solvent portion may not be vitrified well. If the concentration is higher than 20 w / v%, the viscosity may become too high, which may deteriorate the handling properties. For example, the concentration of the polymer or its salt is preferably 5 w / v% or more, and more preferably 10 w / v% or more. Also, the concentration of the polymer or its salt is preferably 50 w / v% or less, and more preferably 20 w / v% or less. Preferably, the concentration of the polymer or its salt is 5 w / v% or more and 20 w / v% or less.

[0118] The concentration of the saccharide or its salt in the cryopreservation solution of the present invention is about 1 w / v% or more and 10 w / v% or less. That is, the ratio of the polymer to the saccharide in the present invention is preferably about 10:1. If the concentration of the saccharide or its salt is less than 1 w / v%, the effect of the present invention may not be sufficiently obtained. In addition, even if the saccharide is added to a concentration of 10 w / v% or more, it is difficult to obtain further effects as a cell protection component.

[0119] Since the cryopreservation solution of the present invention is a non-permeable cryoprotective reagent, the biological sample to be cryopreserved using the cryopreservation solution of the present invention is not particularly limited. It can be used for cryopreservation of various types of cells. In addition, the origin of the cells is not particularly limited. The cryopreservation solution of the present invention can effectively suppress ice crystal formation and recrystallization during freezing and thawing, so it can be used well for mammalian cells with complex structures. Therefore, it can be applied to the cryopreservation of cells of various animal species, including mice, dogs, and humans. Furthermore, the cryopreservation solution of the present invention is known to be more damaged during freezing than general culture cells, and is particularly suitable for cryopreservation of reproductive cells such as stem cells, early embryos, eggs, sperm, and fertilized eggs, which are known to be significantly damaged during freezing compared to general culture cells, and which are inevitably subject to a significant decrease in survival rate by the so-called conventional slow freezing method. In addition, since the cryopreservation solution of the present invention does not contain chemical substances such as DMSO and ethylene glycol that can act as differentiation factors, it can be used to preserve cells that need to be kept undifferentiated. For example, stem cells for regenerative medicine applications can be cryopreserved without the risk of differentiation. It is possible to add chemical substances such as DMSO and ethylene glycol at low concentrations where the above risks are not a problem.

[0120] In addition, because no serum or serum-derived proteins are added, there is no risk of contamination with bacteria or viruses. However, it is possible to add proteins that are free of the risk of infectious diseases.

[0121] In addition, because the cryopreservation solution of the present invention has an excellent effect of stabilizing the glassy state in a frozen state, it can also be used for cryopreservation of large cells such as oocytes and fertilized eggs, which are known to be difficult to preserve, as well as organized cell structures, tissues, and tissue-like materials, such as tissues obtained through regenerative medicine.

[0122] That is, the cryopreservation solution of the present invention can be used for biological samples selected from cells, tissues, or tissue-like substances such as membranes or aggregates, and can achieve a high survival rate. In particular, the cryopreservation solution of the present invention can be advantageously used for cryopreservation of mesenchymal stem cells, hematopoietic stem cells, neural stem cells, somatic stem cells such as bone marrow stem cells and germline stem cells, blood cells, endothelial cells, and the like, regardless of whether the cells are primary cells or established cells, and in particular, for cryopreservation of stem cells of primates, which are considered to have lower freezing resistance than mice, for cryopreservation of tissues for transplantation, and for cryopreservation of germline cells in reproductive medicine.

[0123] In addition, in the present invention, if a polymer that is a biological component is used as the polymer, it is possible to thaw a frozen biological sample and use it as a solution for cell administration. Furthermore, in the case of tissue or tissue-like matter, it is possible to add such a polymer solution of the present invention at the organization stage and freeze-store it as it is. This is thought to reduce problems after transplantation. In such a case, the preferred polymer for the cryopreservation solution of the present invention is hyaluronic acid. In particular, the hyaluronic acid has a viscosity average molecular weight of more than 3000, more preferably more than 5000, and 60000 or less, more preferably 20000 or less.

[0124] The cryopreservation solution of the present invention is a cryopreservation solution for the slow freezing method. That is, in the freezing method using the cryopreservation solution of the present invention, the cooling rate is preferably 10°C / min or less, and more preferably, the cooling rate is about 1°C / min or less. It is considered that at such a cooling rate, the inside of the cells is appropriately dehydrated, the intracellular fluid is satisfactorily vitrified, and a high cell cryoprotection effect can be obtained.

[0125] The cryopreservation method of the present invention includes placing a biological sample in a cryopreservation solution containing, 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 3,000 or less; freezing the cryopreservation solution containing the biological sample; and preserving the biological sample by maintaining the cryopreservation solution containing the biological sample at a temperature of -27°C or less.

[0126] The viscosity average molecular weight of the polymer or its salt used in the cryopreservation method of the present invention is desirably 400,000 or less, particularly 200,000 or less. This is because the viscosity can be adjusted to a low level and the cryopreservation solution is easy to handle.

[0127] The salt of the polymer and the saccharide may be a metal salt, a halogen salt or a sulfate salt of the polysaccharide. The metal salt is preferably an alkali metal or an alkaline earth metal salt. The alkali metal or the alkaline earth metal may be sodium, potassium, calcium or the like. The halogen may be chlorine, bromine or the like. Since the cryopreservation can be performed by freezing the cryopreservation solution containing the biological sample in a deep freezer at -80°C at a cooling rate of 10°C / min or less, preferably at a cooling rate of about 1°C / min or less, and storing it as it is, there is no need for a rapid operation for rapid cooling required in the conventional vitrification method. Therefore, it is considered that the operability is improved and at the same time a stable preservation effect can be obtained.

[0128] The range of the cryopreservation temperature is not limited as long as it is −27° C. or lower, but the upper limit is desirably −70° C. or lower, preferably −80° C. or lower. The lower limit is desirably −196° C. or higher, preferably −150° C. or higher.

[0129] As the solvent for the cryopreservation solution of the present invention, it is preferable to use an aqueous solvent such as water. In particular, it is preferable to use an isotonic solution in which the salt concentration and sugar concentration are adjusted by sodium ions, potassium ions, calcium ions, etc. so that the osmotic pressure is approximately the same as that of body fluids or cell fluids. Specifically, for example, water, physiological saline, physiological saline with a buffer effect such as phosphate buffered saline (PBS), Dulbecco's phosphate buffered saline, Tris buffered saline (TBS), HEPES buffered saline, balanced salt solutions such as Hanks' balanced salt solution, Ringer's solution, lactate Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution, etc. are exemplified, but are not limited to these. In addition, the solvent may contain other optional components such as isotonic agents, chelating agents, and dissolution aids as long as they do not impair the effects of the present invention. In this specification, the term "optional components" means components that may or may not be included. For example, the solvent may be a 5% glucose aqueous solution. In addition, a cell culture medium may be used as the solvent for the cryopreservation solution of the present invention. The culture medium is not particularly limited, and examples thereof include commercially available media, basal media for animal cell culture such as D-MEM, E-MEM, αMEM, RPMI-1640 medium, Ham's F-12, Ham's F-10, and M-199, and general culture media for various cells or tissues. Therefore, the cryopreservation solution of the present invention may be added to the culture solution or cell suspension after cell culture so that the cryopreservation agent reaches a desired concentration.

[0130] In addition, the cryopreservation solution of the present invention may be pH adjusted as necessary. For example, when the hydrophilic group of the monomer having a hydrophilic group is a carboxylic acid group, the cryopreservation solution containing a polymer obtained by polymerizing such a monomer may be acidic. In addition, when a sugar has a carboxylic acid group, the cryopreservation solution may be acidic due to such sugar. In such a case, the cryopreservation solution may be made into a neutral solution by adjusting the pH, which may be more suitable for the survival of the cells to be cryopreserved, and the survival rate of the cells may be improved. There is no limitation on the salt used for adjusting the pH, and any salt commonly used for adjusting the pH of an aqueous solution may be used.

[0131] In the method for freezing and storing biological samples using the cryopreservation solution of the present invention, the vitrified state is stabilized and the toxicity is low, so that the biological sample can be stably stored for a long period of time. In this specification, long-term stable storage means that, for example, the viability of a biological sample, such as cells, after thawing using the cryopreservation solution of the present invention is reduced by less than 10%, preferably less than 5%, after 5 months, or by less than 20%, preferably less than 10%, after 6 months, or by less than 15%, preferably less than 30%, after 12 months, based on the viability of the cells immediately before storage. In addition, in this specification, long-term stable storage means that, for example, when cells are frozen and stored at -80°C for a long period of time, thawed, and then stored at 4°C, even 24 hours after thawing, the viability is reduced by less than 5%, based on the viability of the cells immediately after thawing. It is considered that the cryopreservation solution of the present invention can freeze and store cells under less stressful conditions than DMSO solution. Therefore, the cryopreservation solution of the present invention can obtain a very high cell survival rate after thawing, compared with cryopreservation using a conventional cryopreservation solution such as a 10% DMSO solution. Furthermore, not only cells immediately after thawing, but also cells refrigerated after thawing can exhibit a high cell survival rate. The cryopreservation solution of the present invention can stably cryopreserve cells for a long period of time without changing the properties of the cells. EXAMPLES

[0132] The present invention will be specifically described based on examples, but the present invention is not limited thereto. In the following, the hyaluronic acid manufactured by Lifecore Biomedical is sodium hyaluronate, but for the sake of simplicity, it is described as "hyaluronic acid".

[0133] Cryopreservation agents and fluids <Preparation of test samples and sample solutions> Example 1 In a pressure-resistant container with a volume of 2L, polymeric 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 subcritical processing was carried out at a processing temperature of 175°C, a processing pressure of 0.89MPa, and a processing time of 3 minutes.Then, the subcritical processing product was dried by freeze-drying or spray-drying.As a result, a mixture of polymeric hyaluronic acid with a viscosity average molecular weight of about 10,000 and hyaluronic acid with a viscosity average molecular weight of 1000, that is, the cryopreservation agent of the present invention, was obtained.

[0134] Regarding the viscosity average molecular weight, the hyaluronic acid decomposition product by subcritical treatment obtained in this Example 1 was confirmed by HPLC (Figure 18), and was considered to be a mixture of high molecular weight hyaluronic acid and low molecular weight hyaluronic acid. Therefore, the high molecular weight hyaluronic acid was precipitated in ethanol, and the low molecular weight hyaluronic acid was fractionated in the supernatant. When this fractionated supernatant was reanalyzed by HPLC, it was found to be roughly consistent with the HPLC peak of the hyaluronic acid in Production Example 1 described below (Figure 17A). Therefore, the viscosity average molecular weight of the low molecular weight hyaluronic acid component obtained in this Example 1 was estimated to be 1000.

[0135] The intrinsic viscosity of the high molecular weight hyaluronic acid was 0.49 dL / g, and the viscosity average molecular weight was 10,000.

[0136] 1 g of this test cryopreservation agent was dissolved in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent is water) to obtain the test cryopreservation solution of Example 1 (i.e., the concentration of the hyaluronic acid sample in the test cryopreservation solution is 10 w / v%). Including this Example 1, in the examples, production examples and comparative examples of the present invention, αMEM medium is used as the solvent, but ultrapure water may be used as the solvent instead of αMEM medium. In addition, in the DSC measurement described later, ultrapure water is used as the solvent instead of αMEM. Note that each test sample obtained in the following examples was prepared to a predetermined concentration using an appropriate solvent in the test for each evaluation described later as a cryopreservation agent for the cryopreservation solution and used.

[0137] Manufacturing Example 1 The same procedure as in Example 1 was carried out except that the subcritical treatment was carried out for 7 minutes, to obtain a test sample which was a hyaluronic acid fragment having a viscosity average molecular weight of 1000. The intrinsic viscosity was 0.08 dL / g.

[0138] Manufacturing Example 2 The same procedure as in Example 1 was carried out except that the subcritical treatment was carried out for 5 minutes, to obtain a test sample which was a hyaluronic acid fragment having a viscosity average molecular weight of 2000. The intrinsic viscosity was 0.14 dL / g.

[0139] Manufacturing Example 3 The same procedure as in Example 1 was carried out except that the subcritical treatment was carried out for 4 minutes, to obtain a test sample which was a hyaluronic acid fragment having a viscosity average molecular weight of 3000. The intrinsic viscosity was 0.19 dL / g.

[0140] Comparative Example 1 DMSO (Nacalai Tesque, Inc., cell culture grade) was used as a test sample. 1 mL of this test sample was dissolved in 10 mL of α-MEM medium (Gibco, product number C1257-1500BT, the solvent is water) to obtain a test cryopreservation solution of Comparative Example 1 (i.e., the DMSO concentration of the test sample in the test cryopreservation solution was 10 w / v%).

[0141] Comparative Example 2 The αMEM medium (Gibco, product number C1257-1500BT, the solvent is water) used as a solvent was used as a test sample.

[0142] Comparative Example 3 1 g of gelatin (viscosity average molecular weight 315000, manufactured by Nitta Gelatin Co., Ltd.) was dissolved in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent is water) as a solvent to obtain a test cryopreservation solution of Comparative Example 3 (i.e., the gelatin concentration of the test sample in the test cryopreservation solution was 10 w / v%). The intrinsic viscosity was 0.50 dL / g.

[0143] Comparative Example 4 1g of high molecular weight hyaluronic acid (manufactured by Shanghai Easier Industrial Development Co., Ltd.) with viscosity average molecular weight of 1,000,000 is dissolved in 100mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, solvent is water) as solvent, to obtain the test cryopreservation solution of Comparative Example 4 (i.e., the hyaluronic acid concentration of the test sample in the test cryopreservation solution is 1w / v%).The intrinsic viscosity is 17.2dL / g.

[0144] Comparative Example 5 Hyaluronic acid (lifecore biomedical; powder) with viscosity average molecular weight of 15000 was used as a test sample as a cryopreservation agent. 1g of this test sample was dissolved in 10mL of αMEM medium (Gibco, product number C1257-1500BT, solvent is water) to obtain a test cryopreservation solution of Comparative Example 5 (i.e., the hyaluronic acid concentration of the test sample in the test cryopreservation solution is 10w / v%). The intrinsic viscosity was 0.65dL / g.

[0145] Comparative Example 6 Hyaluronic acid (lifecore biomedical; powder) with viscosity average molecular weight of 50000 was used as the test sample. 1g of this test sample was dissolved in 10mL of αMEM medium (Gibco, product number C1257-1500BT, solvent is water) to obtain the test cryopreservation solution of Comparative Example 6 (i.e., the hyaluronic acid concentration of the test sample in the test cryopreservation solution is 10w / v%). The intrinsic viscosity was 1.67dL / g.

[0146] Comparative Example 7 Hyaluronic acid (lifecore biomedical; powder) with viscosity average molecular weight of 125000 was used as the test sample. 1g of this test sample was dissolved in 10mL of αMEM medium (Gibco, product number C1257-1500BT, solvent is water) to obtain the test cryopreservation solution of Comparative Example 7 (i.e., the hyaluronic acid concentration of the test sample in the test cryopreservation solution is 10w / v%). The intrinsic viscosity was 3.4dL / g.

[0147] Comparative Example 8 Pullulan (Tokyo Chemical Industry Co., Ltd.) with a viscosity average molecular weight of 373,000 was used as the test sample. 1 g of this test sample was dissolved in 10 mL of αMEM medium (Gibco, product number C1257-1500BT, solvent is water) to obtain the test cryopreservation solution of Comparative Example 8 (i.e., the hyaluronic acid concentration of the test sample in the test cryopreservation solution is 10 w / v%). The intrinsic viscosity was 0.55 dL / g.

[0148] Comparative Example 9 Chondroitin sulfate sodium salt A (manufactured by Sigma) with a viscosity average molecular weight of 23000 was used as the test sample. 1 g of this test sample was dissolved in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent was water) to obtain a test cryopreservation solution of Comparative Example 9 (i.e., the chondroitin sulfate concentration of the test sample in the test cryopreservation solution was 10 w / v%). The intrinsic viscosity was 0.97 dL / g.

[0149] Example 2 1 g of the test sample (hyaluronic acid with a viscosity average molecular weight of 15,000) of Comparative Example 5 was dissolved in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent is water) to obtain a test sample solution with a hyaluronic acid concentration of 10 w / v%, to which the test sample (hyaluronic acid fragment sample with a viscosity average molecular weight of 1,000) of Preparation Example 1 was added at a final concentration of 1 w / v% to obtain a test frozen storage solution of Example 2.

[0150] Example 3 The pH of the test cryopreservation solution of Example 2 (containing hyaluronic acid with a viscosity average molecular weight of 15,000 and the test sample of Manufacturing Example 1) was adjusted to neutral using 10 mM Tris-HCl to prepare the test cryopreservation solution of Example 3.

[0151] Example 4 1 g of the test sample of Comparative Example 8 (pullulan with a viscosity average molecular weight of 373,000) was dissolved in 10 mL of α-MEM medium (Gibco, product number C1257-1500BT, the solvent is water) to obtain a test sample solution with a pullulan concentration of 10 w / v%, to which the test sample of Preparation Example 1 (hyaluronic acid fragment sample with a viscosity average molecular weight of 1,000) was added at a final concentration of 1 w / v% to obtain the test frozen storage solution of Example 4.

[0152] Example 5 The pH of the test frozen storage solution of Example 4 (containing pullulan with a viscosity average molecular weight of 373,000 and the test sample of Production Example 1) was adjusted to neutral using 10 mM Tris-HCl to prepare the test frozen storage solution of Example 5.

[0153] Example 6 1 g of the test sample of Comparative Example 8 (pullulan with a viscosity average molecular weight of 373,000) was dissolved in 10 mL of α-MEM medium (Gibco, product number C1257-1500BT, the solvent is water) to obtain a test sample solution with a pullulan concentration of 10 w / v%, to which the test sample of Preparation Example 1 (hyaluronic acid fragment sample with a viscosity average molecular weight of 1,000) was added at a final concentration of 5 w / v% to obtain the test frozen storage solution of Example 6.

[0154] Example 7 The pH of the test frozen storage solution of Example 6 (containing pullulan with a viscosity average molecular weight of 373,000 and the test sample of Production Example 1) was adjusted to neutral using 10 mM Tris-HCl to prepare the test frozen storage solution of Example 7.

[0155] Example 8 1 g of the test sample of Comparative Example 9 (chondroitin sulfate sodium salt with a viscosity average molecular weight of 23,000) was dissolved in 10 mL of α-MEM medium (Gibco, product number C1257-1500BT, the solvent is water) to prepare a test sample solution with a sodium chondroitin sulfate concentration of 10 w / v%, to which the test sample of Production Example 1 (hyaluronic acid fragment sample with a viscosity average molecular weight of 1,000) was added at a final concentration of 1 w / v% to obtain the test frozen storage solution of Example 8.

[0156] Example 9 1 g of the test sample of Comparative Example 5 (hyaluronic acid with a viscosity average molecular weight of 15,000) was dissolved in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent is water) to prepare a test sample solution with a hyaluronic acid concentration of 10 w / v%, to which sucrose (manufactured by Nacalai Tesque, Inc., molecular weight (same value as viscosity average molecular weight) is 342) was added at a final concentration of 1 w / v% to obtain a test frozen storage solution of Example 9.

[0157] Example 10 1 g of the test sample (hyaluronic acid with a viscosity average molecular weight of 15,000) of Comparative Example 5 was dissolved in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent is water) to prepare a test sample solution with a hyaluronic acid concentration of 10 w / v%, to which glucuronic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., molecular weight (simulated as viscosity average molecular weight) 194) was added at a final concentration of 1 w / v% to obtain a test frozen storage solution of Example 10.

[0158] Comparative Example 10 Carboxypolylysine in which 60% of the amino groups were carboxylated (viscosity average molecular weight 13,400: BioVerde, Inc., CryoScarless DMSO free) was used as a cell preservation solution.

[0159] Comparative Example 11 The test sample from Production Example 1 (hyaluronic acid fragment sample with a viscosity average molecular weight of 1000) was added at 1 w / v% to carboxypolylysine (viscosity average molecular weight 13,400: BioVerde Co., Ltd., CryoScarless DMSO free) with 60% of the amino groups carboxylated to prepare a test frozen storage solution.

[0160] <Test Example 1: Cryopreservation of primary human mesenchymal stem cells using a test cryopreservation solution and evaluation of its preservation effect> Cultured primary human mesenchymal stem cells (Lonza PT2501) were added at a concentration of 1 × 10 6 The cells were suspended in the test cryopreservation solutions (serum-free) of Example 1 and Comparative Examples 1, 2, 4, and 5 at a concentration of 1000 cells / mL.

[0161] The cell suspensions containing each test cryopreservation solution were then frozen in a slow cell freezer (Nalgene® Mr. Frosty) in a -80°C freezer. The frozen cell suspensions containing each test sample were stored at -80°C for 7 days and then rapidly thawed in a 37°C warm bath. The cell suspensions containing each test cryopreservation solution after thawing were evaluated for cell viability by trypan blue staining immediately after thawing. The results are shown in Figure 1.

[0162] FIG. 1 shows the cell viability after thawing of cells cryopreserved in a medium without cryopreservation solution, i.e., in the test cryopreservation solution of Comparative Example 2, or in the test cryopreservation solutions of Example 1 and Comparative Examples 1, 4, and 5. As shown in FIG. 1, in Example 1, i.e., when the hyaluronic acid sample obtained by subcritical treatment of hyaluronic acid has a viscosity average molecular weight of about 10,000, and further contains a hyaluronic acid cleavage product having a viscosity average molecular weight of 1000, i.e., a molecular weight smaller than 10,000, contains the test cryopreservation solution of Example 1, which is a cryopreservation agent in the cryopreservation solution, a remarkably high preservation effect exceeding 90% was obtained. The preservation effect here was even higher than that of Comparative Example 1, which contains DMSO, a commonly used cryopreservation agent, as a cryopreservation agent. It can be seen that the cryopreservation solution of the present invention is an excellent cryopreservation solution with low cytotoxicity. In Comparative Example 4, which contains a high molecular weight hyaluronic acid with a molecular weight as large as 1,000,000 as a test sample, almost no cell protection effect was observed. In Comparative Example 2, which does not contain a cryopreservation agent, the cell viability was almost 0%. In addition, in Comparative Example 5, in which the test sample, which is hyaluronic acid with a viscosity average molecular weight of 15,000, is used as the cryopreservation agent, the cell protection effect as high as that of Example 1 was not observed. Therefore, it was confirmed that the cryopreservation agent consisting of a polymer and a saccharide with a molecular weight smaller than that is contained in the cryopreservation solution is effective for improving the cell protection effect of the cryopreservation solution. In addition, since the cryopreservation agent of the present invention shows a high cell preservation effect even when prepared as a cryopreservation solution using a culture solution such as αMEM medium as a solvent, the cryopreservation agent of the present invention can be added directly to the culture solution of cells, and the cells can be suspended and frozen for preservation. It is not necessary to centrifuge the cells to be preserved, and it is considered that a higher cell survival rate and maintenance of cell properties are possible.

[0163] <Test Example 2: Evaluation of cryopreservation solution analysis by differential scanning calorimetry> The test samples of Example 1 and Comparative Examples 2 and 5 were prepared to a final concentration of 10% using ultrapure water instead of αMEM medium, and used as samples for differential scanning calorimetry. Each sample was scanned with a differential scanning calorimeter (DSC) as follows. (1) After holding at 20°C for 1 minute, cool to -80°C at any speed. (2) After holding at -80°C for 1 minute, increase the temperature to 20°C at a rate of 10°C / min.

[0164] The results are shown in Figures 2A and 2B. In Comparative Example 2, in which the test sample was ultrapure water (i.e., a sample that was ultrapure water), only the ice crystal melting peak of free water was observed as shown in Figure 2A. In Comparative Example 5, in which the test sample was hyaluronic acid with a viscosity average molecular weight of 15,000, a shift in the ice crystal melting peak and a drop in the freezing point were observed compared to Comparative Example 2, and a glass transition was confirmed near -20°C (Figure 2B). On the other hand, in the test sample of Example 1, which contains hyaluronic acid with a viscosity average molecular weight of about 10,000 as well as a hyaluronic acid cleavage product (low molecular weight hyaluronic acid with a viscosity average molecular weight of 1000), only a glass transition was confirmed near -23°C ± 4°C (Figure 2B), and the ice crystal melting peak was extremely small and almost not observed. Therefore, it can be seen that when the cryopreservation solution contains a cryopreservation agent consisting of a polymer and a saccharide with a molecular weight smaller than that, it has a significantly advantageous effect of suppressing recrystallization of water during thawing.

[0165] Next, the test samples of Example 1 and Comparative Examples 1 and 2 were adjusted to a final concentration of 10% using ultrapure water instead of αMEM medium to prepare samples for differential scanning calorimetry. Each sample was scanned with a differential scanning calorimeter (DSC) as follows. (1) Hold at 20°C for 1 minute, then cool to -80°C at a rate of 5°C / min. (2) After holding at -80°C for 1 minute, increase the temperature to 20°C at a rate of 10°C / min.

[0166] The results are shown in FIG. 3A (Comparative Example 2), FIG. 3B (Comparative Example 1), and FIG. 3C (Example 1), respectively.

[0167] In Comparative Example 2, in which the test sample is ultrapure water, a very large exothermic peak associated with ice crystal formation was observed during the temperature drop process, as shown in FIG. 3A. Also, a large amount of heat of fusion associated with ice crystal melting was observed during the temperature rise process. In Comparative Example 1, in which the test sample is DMSO, only a small peak associated with ice crystal formation was observed, as shown in FIG. 3B, and almost no peak associated with ice crystal melting was observed. This indicates that the cryopreservation solution containing the test sample (DMSO) of Comparative Example 1 is close to a vitrified state in the frozen state. In the cryopreservation solution containing the test sample of Example 1 (hyaluronic acid with a viscosity average molecular weight of 10,000 containing a cleavage product of hyaluronic acid) as a cryopreservation agent, almost no melting peak was observed, as shown in FIG. 3C, and the peak associated with ice crystal formation was much smaller than the peak observed in Comparative Example 1. Therefore, in the cryopreservation solution containing the test sample of Example 1, almost no ice crystals were formed, and it is considered to be in an extremely good vitrified state.

[0168] <Test Example 3: Evaluation of long-term preservation effect of cryopreservation using a test cryopreservation solution for primary human mesenchymal stem cells> Cultured primary human mesenchymal stem cells (Lonza PT2501) were added at a concentration of 1 × 10 6 The cells were suspended in the test cryopreservation solutions (serum-free) of Example 1 and Comparative Example 1 at a concentration of 1000 / mL. The cell suspensions containing each test cryopreservation solution were then frozen in a slow cell freezer (Nalgene (registered trademark) Mr. Frosty) in a -80°C freezer. The frozen cell suspensions containing each test cryopreservation solution were stored at -80°C for up to 12 months, and then the cell suspensions containing each test cryopreservation solution were removed at the designated storage period and quickly thawed in a 37°C warm bath. The cell viability of the cell suspensions containing each test cryopreservation solution after thawing was evaluated by trypan blue staining immediately after thawing. The results are shown in Figure 4A. The cell suspensions frozen and stored at -80°C for 3 months were thawed and then stored at 4°C for one day or one week. The cell viability of the cell suspensions stored at 4°C was evaluated by trypan blue staining. The cell viability after storage at 4°C was calculated based on the cell viability immediately after thawing (i.e., immediately before storage) as 100%. The results are shown in Figure 4B.

[0169] From the results shown in FIG. 4A, it can be seen that the cryopreservation solution of the present invention shows a high cell viability of 95% or more, which remains almost unchanged even after 5 months of storage. On the other hand, in the cryopreservation solution containing the test sample (DMSO) of Comparative Example 1, a decrease in cell viability was already observed after 2 months of cryopreservation. And, it can be seen that after 3 months, the cell viability in the cryopreservation solution of Comparative Example 1 is below 50%. This result is in contrast to the cryopreservation solution of the present invention, which still maintains a high cell viability even after 3 months. After 6 months of cryopreservation, the cryopreservation solution of the present invention only shows a decrease in viability of less than 10%, while the cryopreservation solution of Comparative Example 1 shows a decrease in cell viability of about 25%. Furthermore, in the cryopreservation solution of the present invention, the cell viability is still maintained high even after 12 months of cryopreservation, and the decrease in cell viability is only less than 15%.

[0170] Therefore, it can be seen that the cryopreservation solution of the present invention enables cells to be cryopreserved stably for a long period of time with a high cell survival rate.

[0171] In addition, when cells stored at -80°C for a long period of time were thawed and then stored at 4°C, the cell viability after one day of storage at 4°C in the cryopreservation solution of the present invention was nearly 100% of the cell viability immediately before storage, whereas in Comparative Example 1, it had decreased to about 60%. Even after one week of storage at 4°C, the cell viability was still 40% or more in the cryopreservation solution of the present invention. This indicates that the cells that survived after thawing in the cryopreservation solution of the present invention were essentially cells that were normally proliferating, and that the cryopreservation solution did not damage the cells during storage, confirming that the cryopreservation solution of the present invention has a very excellent cell preservation effect.

[0172] <Test Example 4: Evaluation of properties after cryopreservation using test samples of primary human mesenchymal stem cells> Cultured primary human mesenchymal stem cells (Lonza PT2501) were added at a concentration of 1 × 10 6The cells were suspended in the test cryopreservation solutions (serum-free) of Example 1 and Comparative Example 1 at a concentration of 100 cells / mL. The cell suspensions containing each test cryopreservation solution were then frozen in a slow cell freezer (Nalgene (registered trademark) Mr. Frosty) in a -80°C freezer. The frozen cell suspensions containing each test cryopreservation solution were cryopreserved at -80°C for six months. After six months, the cell suspensions containing each test cryopreservation solution were rapidly thawed in a 37°C warm bath, washed with αMEM, and then plated at 2.5 x 10 cells / mL in a 6-well plate. 4 After 4 days, the HGF and IL-10 concentrations in the culture medium were quantified. The quantification of HGF and IL-10 was performed using dedicated kits (Quantikine (registered trademark) ELISA Human HGF, catalog number DHG00, manufactured by R&D) and Quantikine (registered trademark) ELISA Human IL-10, catalog number D100B, manufactured by R&D), respectively, according to the procedure in the kit instructions. The results are shown in Figures 5A and 5B.

[0173] As shown in Fig. 5A, in the cryopreservation in the presence of the cryopreservation solution of Comparative Example 1 containing DMSO as a cryopreservation agent, the HGF production in cells after thawing was high. On the other hand, in the cryopreservation using the cryopreservation solution of the present invention containing hyaluronic acid with a viscosity average molecular weight of 10000 including hyaluronic acid cleavage product as a cryopreservation agent, the HGF production in cells after thawing was low, being 1 / 3 or less than that of Comparative Example 1. The results in the presence of DMSO show that cells were in a stressed state when stored in the presence of DMSO. Since such high HGF production is not observed when cells are stored in the presence of the cryopreservation agent of the present invention, it can be seen that cells are stably protected in the cryopreservation solution of the present invention.

[0174] 5B, the amount of IL-10 produced was high in the cells cryopreserved using the cryopreservation solution of Example 1, and was very low in the cells cryopreserved using the cryopreservation solution of Comparative Example 1. This result demonstrates that the cryopreservation solution of the present invention can satisfactorily cryopreserve cells while maintaining their functions.

[0175] Since the cryopreservation solution of the present invention does not contain cytotoxic compounds such as DMSO and ethylene glycol, the cryopreservation solution of the present invention has the remarkable effect of being capable of cryopreserving biological samples while maintaining their properties without exposing them to stressful conditions, unlike conventional cryopreservation solutions.

[0176] In addition, because serum or serum-derived proteins are not added, there is no contamination by bacteria or viruses. It is possible to add compounds that have cytotoxicity at low concentrations that do not impair the function of cells. It is also possible to add proteins that are not susceptible to infectious diseases.

[0177] <Test Example 5: Evaluation of properties (undifferentiated state) after cryopreservation using test samples of primary human mesenchymal stem cells> Cultured primary human mesenchymal stem cells (Lonza PT2501) were added at a concentration of 1 × 10 6 The cells were suspended in the test cryopreservation solutions (serum-free) of Example 1 and Comparative Example 1 at a concentration of 1000 / mL. The cell suspensions containing each test cryopreservation solution were then frozen in a slow cell freezer (Nalgene (registered trademark) Mr. Frosty) in a -80°C freezer. The frozen cell suspensions containing each test cryopreservation solution were cryopreserved at -80°C for 6 months. After 6 months, the cell suspensions containing each test cryopreservation solution were removed and rapidly thawed in a 37°C warm bath. The expression intensities of CD90, CD44, and CD105 in the cell suspensions containing each test cryopreservation solution after thawing were analyzed by flow cytometry. The results are shown in Figure 6.

[0178] CD90, CD44, and CD105 are typical surface proteins expressed on mesenchymal stem cells in an undifferentiated state, and are used as undifferentiated markers for mesenchymal stem cells. As shown in FIG. 6, the expression of all undifferentiated biomarkers, CD90, CD44, and CD105, was higher in cells cryopreserved with the cryopreservation solution of Example 1 than in cells cryopreserved with the cryopreservation solution of Comparative Example 1. Therefore, it can be seen that the cells cryopreserved with the cryopreservation solution of Example 1 maintain the expression of undifferentiated markers. That is, the cells cryopreserved with the cryopreservation solution of Example 1 can maintain an undifferentiated state. It can be seen that the cryopreservation of the present invention can reduce the influence on the differentiated state seen in the case of preservation with the cryopreservation solution of Comparative Example 1.

[0179] These results demonstrate that the cryopreservation method using the cryopreservation solution of the present invention can also be suitably applied to the cryopreservation of stem cells, for which it is important to cryopreserve them in an undifferentiated state.

[0180] <Test Example 6: Evaluation of cryopreservation solutions containing various polysaccharides> Cultured primary canine mesenchymal stem cells (cyagen C160) were added at 1 × 10 6 The cells were suspended in the test cryopreservation solutions (serum-free) of Examples 1 to 8 and Comparative Examples 5, 8 and 9 at a concentration of 10000 cells / mL (however, the concentration of the hyaluronic acid sample in the test cryopreservation solution of Example 1 was 20 w / v%). Then, the cell suspensions containing each test cryopreservation solution were frozen in a slow cell freezer (Nalgene (registered trademark) Mr. Frosty) in a -80°C freezer. After one day of cryopreservation, the cell suspensions containing each test cryopreservation solution were taken out and rapidly thawed in a 37°C warm bath. The cell viability of the cell suspensions containing each test cryopreservation solution after thawing was evaluated by trypan blue staining. The results are shown in FIG. 7.

[0181] As shown in FIG. 7, the polymer, which may be any polymer, not limited to hyaluronic acid, and contains a monomer having a hydrophilic group as a repeating unit and has a predetermined molecular weight, was used as a cryopreservation agent in combination with a saccharide or its salt having a smaller molecular weight than the polymer. When the cryopreservation solution prepared by adding a saccharide or its salt is acidic, a higher cell protection effect was obtained by neutralizing the cryopreservation solution by adjusting the pH. It is considered that the pH adjustment provides more suitable conditions for cell survival. In addition, in order for the added saccharide or its salt to act as a cell protection component and further improve the cell survival rate of cells after freeze-drying, it was necessary for the saccharide or its salt to be added to the cryopreservation solution in an amount of 1 to 5 w / v% relative to the test sample, which is a polymer. On the other hand, even if the amount of the added saccharide or its salt was increased to more than 10 mass% relative to the test sample and the content in the cryopreservation solution was further increased, no further effect on the improvement of cell survival rate was observed.

[0182] Furthermore, the results shown in FIG. 7 demonstrate that the cryopreservation solution of the present invention exhibits a favorable cryoprotective effect not only on human mesenchymal stem cells, but also on canine mesenchymal stem cells.

[0183] <Test Example 7: Evaluation of saccharides having a viscosity average molecular weight of 3000 or less as a cell protective component> Cultured primary canine mesenchymal stem cells (cyagen C160) were added at 1 × 10 6 The cells were suspended in the test cryopreservation solutions (serum-free) of Examples 9, 10 and 2 at a concentration of 1000 / mL. The cell suspensions containing each test cryopreservation solution were then frozen in a slow cell freezer (Nalgene (registered trademark) Mr. Frosty) in a -80°C freezer. After one day of cryopreservation, the cell suspensions containing each test cryopreservation solution were removed and rapidly thawed in a 37°C warm bath. The cell viability of the thawed cell suspensions containing each test cryopreservation solution was evaluated by trypan blue staining immediately after thawing. The results are shown in Figure 8.

[0184] As shown in Fig. 8, compared with Comparative Example 5 (Fig. 1) containing only hyaluronic acid with a viscosity average molecular weight of 15000 as a cryopreservation agent, when saccharides or salts thereof having a molecular weight smaller than that of hyaluronic acid with a viscosity average molecular weight of 15000 are used in combination with hyaluronic acid with a viscosity average molecular weight of 15000 as a cryopreservation agent, the cell protection effect is improved. When sucrose, which is sometimes added to conventional cryopreservation solutions from the viewpoint of water retention, is added to the polymer cryopreservation agent (Example 9), the cell protection effect is also improved, but it can be seen that a test sample that is glucuronic acid (Example 10) and / or the hyaluronic acid fragments of Production Example 1 (Example 2) containing fragments obtained by subcritical treatment and having a viscosity average molecular weight of 1000, is added to the polymer as a cell protection component, that is, as saccharides or salts thereof having a viscosity average molecular weight of 3000 or less according to the present invention, a significantly higher cell protection effect can be obtained. These results indicate that cytoprotective components that are particularly preferred are cleavage products of glycosaminoglycans and / or sugars that are constituent monosaccharides of glycosaminoglycans, in particular sugars that are cleavage products of hyaluronic acid and glucuronic acid, a constituent monosaccharide of hyaluronic acid.

[0185] <Test Example 8: Evaluation of cells in frozen cryopreservation solution (evaluation of intracellular vitrification state)> Cultured primary canine mesenchymal stem cells (cyagen C160) were added at 1 × 10 6 The cells were suspended in the test cryopreservation solutions (serum-free) of Example 1 and Comparative Example 1 at a concentration of 100000000000000 / mL. As a control, a control suspension was prepared in the same manner by suspending the cells in the test sample solution of Comparative Example 2 consisting of αMEM medium without a cryopreservative. Then, 5 μL of the cell suspensions and the control suspensions containing each test cryopreservation solution were added to a hard glass sample placement plate (16φ×0.12 mm), covered with a hard glass cover glass (12φ×0.12 mm), cooled to −80° C. at 5° C. / min on a linKam microscope cooling stage (THMS600), and images were taken at −80° C. with a transmitted light microscope (Olympus BX53). The results are shown in FIGS. 9A-C.

[0186] FIG. 9A shows the results for the control suspension, and it can be seen that in the medium alone, ice crystals are generated within the cells, causing light to be diffusely reflected, causing the cells to turn dark. FIG. 9B shows the results for Comparative Example 1, in which DMSO is used as the test sample. In FIG. 9B, it can be seen that the cells also turn dark, and that minute ice crystals have formed. FIG. 9C shows the results for the cryopreservation solution containing the test sample of Example 1. It can be seen that the cells turn bright, and that the inside of the cells has been vitrified into an amorphous state.

[0187] From this result, it can be seen that the cryopreservation solution of the present invention freezes cells in a vitrified state.And it can be seen that by containing sugars that are the cleavage products of hyaluronic acid, the glass state can be formed more stably.It is considered that the sugars with a viscosity average molecular weight of 3000 or less suppress the formation of ice crystals around cells, so that vitrification occurs stably and efficiently.

[0188] <Test Example 9: Evaluation of intracellular vitrification state using brightness difference> Cultured primary canine mesenchymal stem cells (cyagen C160) were added at 1 × 10 6 The cells were suspended in the test cryopreservation solutions (serum-free) of Example 1 and Comparative Example 1 at a concentration of 10 ...

[0189] The difference in brightness (absolute value) between the inside and outside of the cell in the observation image shown in Figure 10A was analyzed using the image analysis software ImageJ (https: / / imagej.nih.gov / ij / ). In each image, the brightness of the solvent region, the brightness of the inside of the cell, and the Munsell brightness were read under the same conditions, and the read data of the brightness of the solvent region and the brightness of the inside of the cell were compared with the read data of each Munsell brightness. The Munsell brightness of the closest read data was adopted as the brightness of the solvent region and the brightness of the inside of the cell, and the brightness of the solvent and the brightness of the inside of the cell were quantified (Munsell valued). Note that if the read value of the brightness of the intracellular region or solvent region read by the image analysis software is darker than the read value of the darkest Munsell brightness value 0, the Munsell value of the intracellular region or solvent region is set to 0 for convenience, and if the read value of the brightness of the intracellular region or solvent region read by the image analysis software is brighter than the read value of the brightest Munsell brightness value 10, the Munsell brightness of the intracellular region or solvent region is set to 10 for convenience.

[0190] As a result of the measurements, in the control suspension (Comparative Example 2), the Munsell value of the solvent region was 5, the Munsell value of the intracellular region was 0, and the Munsell value difference between the solvent region and the intracellular region was 5. In Comparative Example 1, the value of the solvent region was 4, the Munsell value of the intracellular region was 0, and the Munsell value difference was 4. In contrast, in Example 1, the Munsell value of the solvent region was 4, the Munsell value of the intracellular region was 2, and the Munsell value difference was 2 (FIG. 10B).

[0191] That is, in Example 1, the brightness of the intracellular region and the brightness of the solvent region are close to each other. This result indicates that in the cryopreservation solution containing the test sample of Example 1, the frozen cells turn bright, that is, the inside of the cells are vitrified. It is desirable that the brightness difference between the intracellular region and the solvent region is 3 or less. It is also desirable that the brightness value of the solvent region is greater than or equal to the brightness value of the intracellular region.

[0192] <Test Example 10: Evaluation of cell area in frozen cryopreservation solution> Cultured primary canine mesenchymal stem cells (cyagen C160) were added at 1 × 10 6 The cells were suspended in the test cryopreservation solutions (serum-free) of Example 1 and Comparative Example 1 at a concentration of 1000 cells / mL. As a control, a control suspension was prepared in the same manner by suspending the cells in a medium (i.e., medium only) containing a test sample solution of Comparative Example 2, which is made of αMEM medium without a cryopreservative. Then, the cell suspensions containing each test cryopreservation solution and the control suspension were cooled to -80°C at 5°C / min on a linKam microscope cooling stage (THMS600), and then rapidly thawed at -50°C / min. The area of ​​10 cells present in the field of view before cooling and after thawing was calculated using image analysis software ImageJ. The results are shown in FIG. 11.

[0193] As shown in Figure 11, in Comparative Example 2, which is a medium only, and Comparative Example 1, which is a test sample containing DMSO, the area of ​​the cells increased after freezing, while after freezing using the cryopreservation solution of the present invention, the cells were reduced to about 1 / 3. It was confirmed that the inside of the cells was well dehydrated during freezing. It is desirable that the cells be reduced to less than 1 / 1 and more than 1 / 3.5 of their orthogonal projection area in the medium.

[0194] <Test Example 11: Evaluation of cryoprotection for various cells> Cultured mouse-derived macrophage-like cell line (RAW264), human colon carcinoma-derived cells (Caco-2), and primary human pulmonary microvascular endothelial cells (HMVEC) were each cultured at 1 × 10 6 The cells were suspended in the test cryopreservation solutions (serum-free) of Example 1 and Comparative Example 1 at a concentration of 10 ...

[0195] 12, when cryopreserved using the cryopreservation solution of the present invention, all cells obtained had a high cell viability that was almost equal to or higher than that of Comparative Example 1, in which DMSO was used as the test sample. This result confirmed that the cryopreservation solution of the present invention can cryopreserve various types of cells with high cell viability, regardless of whether they are primary cells or established cells, and regardless of the cell origin.

[0196] <Test Example 12: Evaluation of the effect of the saccharide of the present invention on cell viability> Cultured primary canine mesenchymal stem cells (cyagen C160) were added at 1 × 10 6 The cells were suspended in each of the following test cryopreservation solutions (serum-free) at a concentration of 10 ...

[0197] Fig. 13 shows the effect of adding a test sample, which is the hyaluronic acid fragment of Production Example 1, containing a fragment obtained by subcritical treatment and having a viscosity average molecular weight of 1000, to polymers of different molecular weights. The test cryopreservation solutions used were the test cryopreservation solution of Example 1, the test cryopreservation solution of Comparative Example 6, and the test sample of Production Example 1 (hyaluronic acid fragment sample with a viscosity average molecular weight of 1000) added to the test cryopreservation solution of Comparative Example 6 in an amount of 1 w / v %.

[0198] In the experiment using Comparative Example 6, in which the viscosity average molecular weight of hyaluronic acid is 50000, the viscosity of the test cryopreservation solution was high, and there was some difficulty in handling, such as foaming during the preparation of the cell suspension, but it was possible to prepare the concentration of the cryopreservation solution in the cell suspension to 10w / v%.As shown in Figure 13, the cryopreservation solution of Example 1, in which the polymer having a viscosity average molecular weight of more than 3000 and less than 500000, the polymer or its salt containing a monomer having a hydrophilic group as a repeating unit, is hyaluronic acid with a viscosity average molecular weight of 10000, and the sugar or its salt having a viscosity average molecular weight of 3000 or less is the sugar that is a cleavage product of hyaluronic acid, had the highest survival rate.Even in Comparative Example 6, in which the viscosity average molecular weight of the polymer is 50000, the cell survival rate was improved by adding Production Example 1, which is a sugar with a viscosity average molecular weight of 1000 that contains a cleavage product of hyaluronic acid.

[0199] 14 shows the results of examining the cell survival rate after thawing, where the cell suspension was frozen under conditions in which the content of the cryopreservation solution in the cell suspension was reduced to result in a low content of polymer as a cryopreservation agent. The cell suspensions with a low content of polymer as a cryopreservation agent were each prepared by suspending cells in a test cryopreservation solution containing 1 w / v% of the test cryopreservation solution of Comparative Example 6, a test cryopreservation solution containing 1 w / v% of the test cryopreservation solution of Comparative Example 6 to which Production Example 1 was added in an amount of 1 w / v%, a test cryopreservation solution containing 1 w / v% of the test cryopreservation solution of Comparative Example 7, and a test cryopreservation solution containing 1 w / v% of the test cryopreservation solution of Comparative Example 7 to which Production Example 1 was added in an amount of 1 w / v%.

[0200] The results in Figure 14 show that when the polymer concentration is approximately 1 w / v%, the effects of the present invention cannot be obtained even when sugars (Production Example 1 containing hyaluronic acid cleavage products with a viscosity average molecular weight of 1000) are added.

[0201] 15 shows the results of investigating the effect of the molecular weight of a component as a saccharide or its salt having a viscosity-average molecular weight of 3000 or less in a cryopreservation solution on cell viability. The test used a test sample solution of Comparative Example 5 containing a test sample with a viscosity-average molecular weight of 15000, and a test cryopreservation solution containing 1 w / v % of a test sample prepared by adding 1 mass % of each of Preparations 1 to 3 (i.e., test samples that are hyaluronic acid fragments with viscosity-average molecular weights of 1000, 2000, and 3000, respectively) to the test sample of Comparative Example 5.

[0202] The results in Figure 15 show that by adding a saccharide or its salt having a smaller molecular weight than the polymer to the cryopreservation agent and then to the cryopreservation solution, a more improved cell survival effect can be obtained, but the effect gradually decreases as the molecular weight of the saccharide increases.

[0203] 16 shows the cell survival rate after thawing when only saccharides or their salts having a molecular weight smaller than that of polymers were used as test samples and were dissolved in ultrapure water to a concentration of 10 w / v % for cryopreservation. The test samples used were those from Production Examples 1 to 3 with viscosity average molecular weights of 1000, 2000, and 3000, respectively.

[0204] As shown in Figure 16, the cell survival effect cannot be obtained only by sugars or their salts having a molecular weight smaller than that of polymers. In Production Example 3, a cell survival rate of about 10% was observed, which is presumably due to hyaluronic acid having a molecular weight larger than 3000 that may be contained in the test sample of Production Example 3.

[0205] <Test Example 13: HPLC analysis of subcritically treated hyaluronic acid samples> A 1 wt% aqueous solution of the test samples of Preparation Examples 1 to 3 was prepared and filtered through a 0.45 μm membrane filter (Millipore), and the components of each test sample were analyzed by HPLC. As the mobile phase, Solution A: 16 mM NaH2PO4 aqueous solution, Solution B: 800 mM NaH2PO4 aqueous solution, were used, and the components were separated using a ZORBAX NH2 (Agilent Technologies, Inc., column size φ4.6 × 250 mm, particle size 5 μm) normal phase HPLC column at a flow rate of 1.0 mL / min, a column temperature of 40 °C, and a detection wavelength of 210 nm. The gradient conditions were mobile phase B concentration 0% (0 min) → mobile phase B concentration 100% (60 min). The results are shown in Figures 17A to C. In addition, as standards, aqueous solutions containing 0.2 wt% of hyaluronic acid oligosaccharides, HA02 (disaccharide), HA04 (tetrasaccharide), HA06 (hexasaccharide), HA08 (octasaccharide), and HA10 (decasaccharide) (all manufactured by Iduron Co., Ltd.), were prepared and analyzed by HPLC in the same manner. The results are shown in Figure 17D.

[0206] Under these HPLC analysis conditions, monosaccharides are observed at a retention time of 3.5 minutes, and disaccharides are observed at a retention time of around 9 minutes, as shown in Figure 17D. Figure 17A shows the analysis results of a test sample containing the hyaluronic acid cleavage product of Production Example 1 and having a viscosity average molecular weight of 1000, and a peak corresponding to monosaccharides is observed at a retention time of around 3.5 minutes, and a peak corresponding to disaccharides is observed at a retention time of around 9 minutes. In other words, the test sample of Production Example 1 contains such monosaccharide and disaccharide components, which are thought to contribute to the effect of improving cell viability.

[0207] <Test Example 14: HPLC analysis of the test sample of Example 1> The test sample of Example 1 (a hyaluronic acid sample having a viscosity average molecular weight of about 10,000 obtained by subcritical treatment) was subjected to HPLC analysis under the same conditions as those for the HPLC analysis of the test samples of the above-mentioned Production Examples 1 to 3. The results are shown in FIG.

[0208] Similarly, when compared with Fig. 17D, peaks corresponding to monosaccharides and disaccharides are observed at retention times of about 3.5 minutes and about 9 minutes in the test sample of Example 1. In other words, the test sample of Example 1 also contains such monosaccharide and disaccharide components, which is considered to be the reason for the high cell viability effect.

[0209] <Test Example 15: Evaluation of cell viability using a test sample without a polysaccharide structure> The cell viability after cryopreservation was evaluated using the test cryopreservation solution containing gelatin of Comparative Example 3. Cultured primary canine mesenchymal stem cells (cyagen C160) were cultured at 1 × 10 6 The cells were suspended in the test cryopreservation solutions (serum-free) of Comparative Examples 1 and 3 at a concentration of 10 ...

[0210] 19, when gelatin was used as the cryopreservation agent, only a low cell viability rate was obtained compared to Comparative Example 1, in which DMSO was used as the cryopreservation agent. Therefore, it can be seen that the high cell viability rate effect of the cryopreservation solution of the present invention can be obtained by including, in addition to a polymer, a saccharide or a salt thereof having a molecular weight smaller than that of the polymer in the cryopreservation solution.

[0211] <Test Example 16: Evaluation of cell viability by carboxylated polylysine> Cultured primary human mesenchymal stem cells (Lonza PT2501) were added at a concentration of 1 × 10 6 The cells were suspended in the test cryopreservation solutions of Comparative Examples 10 and 11 at a concentration of 1000 / mL.

[0212] The cell suspensions containing each test cryopreservation solution were then frozen in a slow cell freezer (Nalgene® Mr. Frosty) in a -80°C freezer. The cell suspensions containing the frozen test samples were stored at -80°C for 7 days and then rapidly thawed in a 37°C warm bath. The cell viability of the thawed cell suspensions containing each test cryopreservation solution was evaluated by trypan blue staining immediately after thawing. The results are shown in Figure 20.

[0213] The cell viability was 65% in Comparative Example 10. The cell viability was 60% in Comparative Example 11, which was a mixture of carboxypolylysine and Production Example 1. In the case of carboxypolylysine, no effect was observed even when low molecular weight hyaluronic acid was added.

[0214] In carboxypolylysine, the amino groups of polylysine are modified with carboxylic acid to be hydrophobic, and it is presumed that this makes it difficult for water around cells to be replaced by low-molecular-weight hyaluronic acid.

[0215] From the above results, it can be seen that the cryopreservation solution for biological samples containing a polymer and a saccharide of the present invention has a remarkable effect of being able to cryopreserve biological samples with high cell viability by stably vitrifying the inside of cells, basically without the need for the addition of cell-permeable and cytotoxic compounds such as DMSO and ethylene glycol, and / or serum or serum-derived proteins, etc. Cells are well protected and their properties are maintained.

Claims

1. In the 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 3000 or less; A cryopreservation solution for biological samples comprising:

2. The cryopreservation solution according to claim 1, wherein the hydrophilic group of the monomer having a hydrophilic group is at least one selected from the group consisting of a hydroxyl group, a carboxylic acid group and a salt thereof.

3. 3. The cryopreservation solution according to claim 1, wherein the polymer further comprises, as a repeating unit, a nitrogen-containing monomer having an optionally substituted amino group or an optionally substituted amide group.

4. The cryopreservation solution according to claim 3 , wherein the polymer is an alternating copolymer of the monomer having the hydrophilic group and the nitrogen-containing monomer.

5. The cryopreservation solution according to any one of claims 1 to 4, wherein the monomer having a hydrophilic group has a hydroxyl group substituted at an equatorial position.

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

7. The method of any one of claims 1 to 6, wherein the polymer comprises a plurality of sugar residues.

8. The method according to any one of claims 1 to 7, wherein the saccharide is a monosaccharide, a disaccharide, or an oligosaccharide.

9. The freezing storage solution according to claim 8, wherein the sugar is glucose, fructose, galactose, or uronic acid in which the alcohol group of these sugars is oxidized, or an amino sugar in which the alcohol group is replaced with an amino group, sucrose, a cleavage product of a glycosaminoglycan, a constituent monosaccharide of a glycosaminoglycan, or a polymer or combination thereof.

10. The freezing storage medium according to claim 9, wherein the saccharide is glucuronic acid or N-acetylglucosamine.

11. The cryopreservation solution according to any one of claims 1 to 10, wherein the concentration of the polymer in the cryopreservation solution is 5 w / v % or more and 20 w / v % or less.

12. The cryopreservation solution according to any one of claims 1 to 11, wherein the concentration of the sugar in the cryopreservation solution is 1 w / v % or more and 10 w / v % or less.

13. The cryopreservation solution according to any one of claims 1 to 12, which is an intracellular non-permeable cryopreservation solution.

14. The cryopreservation solution according to any one of claims 1 to 13, wherein the biological sample is a cell, a tissue, or a tissue-like substance that is a membrane or an aggregate.

15. The cryopreservation solution according to claim 14, wherein the biological sample is a mesenchymal stem cell, a blood cell, an endothelial cell, or a tissue for transplantation.

16. The cryopreservation solution according to claim 15, wherein the biological sample is a sperm, an egg, or a fertilized egg.

17. A method for freezing a biological sample, comprising the steps of: cooling and freezing the biological sample by a slow freezing method at a cooling rate of 10° C. / min or less using the cryopreservation solution according to any one of claims 1 to 16.

18. A step of placing a biological sample in a cryopreservation solution containing a polymer or a salt thereof having a viscosity average molecular weight of more than 3,000 and not more than 500,000, the polymer 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 3,000 or less; subjecting the cryopreservation solution containing the biological sample to freezing; preserving the biological sample by maintaining the cryopreservation solution containing the biological sample at a temperature of −27° C. or lower; A method for freezing and preserving a biological sample, comprising:

19. 20. The method of claim 18, wherein protection of the biological sample occurs during storage.

20. 20. The method for cryopreserving a biological sample according to claim 18 or 19, wherein the step of placing the biological sample in the cryopreservation solution is carried out before cooling the biological sample.

21. The method for cryopreserving a biological sample according to any one of claims 18 to 20, wherein the biological sample is a cell, a tissue, or a tissue-like substance which is a membrane or an aggregate.

22. The method for freezing and preserving a biological sample according to claim 21, wherein the biological sample is a sperm, an egg, or a fertilized egg.

23. 1. A method for preserving a biological sample, comprising: 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, and a saccharide having a viscosity average molecular weight of 3,000 or less, or a salt thereof, the saccharide being stored at −27° C. or less; A method for preserving a biological sample, characterized in that the preservation is such that, when the biological sample is stored in the presence of the polymer or its salt and the saccharide or its salt for a period of at least 5 months, the biological sample shows a decrease in viability of less than 5% based on the viability of the biological sample immediately before storage.

24. The method for preserving a biological sample according to claim 23, characterized in that the preservation is such that, when the biological sample is stored in the presence of the polymer or its salt and the saccharide or its salt for a period of at least six months, the biological sample shows a decrease in viability of less than 10% based on the viability of the biological sample immediately before preservation.

25. The method for preserving a biological sample according to claim 23, characterized in that when the biological sample is thawed after frozen storage at -27°C or below and then stored at 4°C for one day, the loss in viability is less than 5% based on the viability of the biological sample immediately after thawing.

26. The method for preserving a biological sample according to any one of claims 23 to 25, wherein the biological sample is a cell.

27. 27. The method for preserving a biological sample according to claim 26, wherein the biological sample is a mammalian cell.

28. 28. The method for preserving a biological sample according to claim 27, wherein the biological sample is a mammalian mesenchymal stem cell, a mammalian blood cell, or a mammalian endothelial cell.

29. Use of a mixture of a 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, or a salt thereof, and a saccharide having a viscosity average molecular weight of 3,000 or less, or a salt thereof, for preserving a biological sample at a temperature of -27°C or less.

30. A use of a mixture of 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 monomer having a hydrophilic group as a repeating unit, and a saccharide or its salt having a viscosity average molecular weight of 3,000 or less, for cryopreserving a biological sample.

31. A cryopreservation agent for biological samples, comprising a mixture of 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 monomer having a hydrophilic group as a repeating unit, and a saccharide or its salt having a viscosity average molecular weight of 3,000 or less.

32. The method for freezing and preserving a biological sample according to claim 31, wherein the polymer comprises a pentose, a hexose, or a uronic acid, or a combination thereof, as a repeating unit.

33. The cryopreservation agent for a biological sample according to claim 31 or 32, wherein the polymer further comprises an amino sugar as a repeating unit.

34. The cryopreservation agent for a biological sample according to any one of claims 31 to 33, wherein the hydrophilic group of the polymer is unmodified.

35. The method for freezing and preserving a biological sample according to any one of claims 31 to 34, wherein the polymer is hyaluronic acid, dextran, pullulan, or chondroitin sulfate.

36. The method for freezing and preserving a biological sample according to any one of claims 31 to 35, wherein the saccharide is a monosaccharide, a disaccharide, or an oligosaccharide.

37. The method for freezing and preserving a biological sample according to any one of claims 31 to 36, wherein the sugar is glucose, fructose, galactose, or uronic acid in which the alcohol group of these sugars is oxidized, or an amino sugar in which the alcohol group is substituted with an amino group, sucrose, a cleavage product of a glycosaminoglycan, a constituent monosaccharide of a glycosaminoglycan, or a polymer or combination thereof.

38. A method for producing a cryopreservation solution for a biological sample, comprising: 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; and a saccharide having a viscosity average molecular weight of 3,000 or less, or a salt thereof, A method for producing a cryopreservation solution for biological samples, comprising dissolving a polysaccharide or a salt thereof having a molecular weight of more than 500,000 in water, and then performing an extraction treatment under subcritical conditions of water.

39. The method for producing a cryopreservation solution for biological samples according to claim 38, wherein the subcritical conditions for water are a temperature of 150 to 350° C. and a pressure of 0.5 to 25 MPa.

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