Cryopreservation medium

Abstract

To provide cryopreservation liquids for biological samples with high cell viability, to provide cryopreservation methods for biological samples, and to provide methods for stably storing biological samples for a long period of time.SOLUTION: Provided are a cryopreservation liquid for biological samples, which has a viscosity average molecular weight of more than 3000 and 500000 or less in a solvent, and contains a polysaccharide containing a six-membered ring structure in its main chain or a salt thereof; a cryopreservation method comprising including a biological sample in a cryopreservation liquid containing a polysaccharide or a salt thereof; a method for storing a biological sample; and a cryopreservation agent for biological samples consisting of a polysaccharide or a salt thereof.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 methyl 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 actual molecular weight of the hydrogel described in Patent Document 2 as a 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. In addition, Patent Document 3 discloses fructan, which is a five-membered ring structure, as an active ingredient of a cell preservation solution. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 63-216476 [Patent Document 2] International Publication No. 2016 / 076317 [Patent Document 3] JP 2012-235728 A [Non-patent literature]

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

[0008] 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.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] The cryopreservation solution described in Patent Document 3 requires a high concentration of 20% or more for the active ingredient fructan, and the concentration cannot be adjusted over a wide range.

[0013] 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.

[0014] The present invention has been made in view of the above problems, and provides a cryopreservation solution for appropriately preserving biological samples. In particular, the present invention aims to provide a cryopreservation solution and a cryopreservation method that enable stable long-term preservation of biological samples 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 preserving biological samples. [Means for solving the problem]

[0015] The present invention relates to a cryopreservation solution for biological samples, which contains a polysaccharide or a salt thereof having a viscosity average molecular weight of more than 3000 and not more than 500,000 and containing a six-membered ring structure in the main chain in a solvent. The salt of the polysaccharide 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 the alkaline earth metal may be selected from sodium, potassium, calcium, etc. As the halogen, chlorine, bromine, etc. may be used. The polysaccharide or the salt thereof having a viscosity average molecular weight of more than 3000 and not more than 500,000 and containing a six-membered ring structure in the main chain is preferably contained as a main component in the cryopreservation solution. In this specification, the main component refers to the component having the highest weight ratio among the components dissolved in the solvent. Components other than the main component are secondary components.

[0016] In the present invention, the viscosity average molecular weight of the polysaccharide or its salt containing a six-membered ring structure in the main chain 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 frozen storage solution.

[0017] In the present invention, dextran is preferably excluded from the polysaccharides as the main component. In addition, the cell cryopreservation solution of the present invention may contain dextran as a minor component, such as a cell protective agent, rather than as a main component.

[0018] Cryopreservation media for biological samples that contain polysaccharides containing pentose, hexose or uronic acid or combinations thereof as repeating units are preferred.

[0019] A cryopreservation medium for biological samples that contains polysaccharides that contain amino sugars as repeating units is preferred.

[0020] The functional groups of the polysaccharide or its salt used in the cryopreservation solution for biological samples of the present invention are preferably unmodified.

[0021] Furthermore, as the polysaccharide contained in the cryopreservation solution for biological samples, sulfated polysaccharides can be used.

[0022] A cryopreservation solution for biological samples containing a polysaccharide or a salt thereof having a viscosity average molecular weight of 10,000 or more is preferred.

[0023] A cryopreservation medium for biological samples that contains a polysaccharide that is hyaluronic acid or pullulan is preferred.

[0024] A cryopreservation solution for biological samples in which the concentration of the polysaccharide or a salt thereof is 5 w / v % or more and 20 w / v % or less is preferred.

[0025] Cryopreservation solutions for biological samples that are non-intercellular-permeating cryopreservation solutions are preferred.

[0026] 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.

[0027] 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.

[0028] The freezing preservation medium is preferably for a biological sample, the biological sample being a sperm, an egg, or a fertilized egg.

[0029] 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.

[0030] The present invention also relates to a method for cryopreserving a biological sample, the method comprising the steps of: placing a biological sample in a cryopreservation solution containing a polysaccharide or a salt thereof, the polysaccharide or salt having a viscosity-average molecular weight of more than 3000 and not more than 500000 and containing a six-membered ring structure in the main chain, in a solvent; 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 lower. The salt of the polysaccharide 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 sodium, potassium, calcium, or the like. The halogen may be chlorine, bromine, or the like.

[0031] The viscosity average molecular weight of the polysaccharide or its salt containing a six-membered ring structure in the main chain 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, because the viscosity can be adjusted to a low level and the solution is easy to handle as a cryopreservation solution.

[0032] 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.

[0033] In the method for cryopreserving a biological sample of the present invention, it is desirable that the polysaccharide in the cryopreservation solution is a polysaccharide other than dextran.

[0034] In the method for cryopreserving a biological sample, the step of placing the biological sample in the cryopreservation solution is preferably carried out before cooling the biological sample.

[0035] 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.

[0036] 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.

[0037] The present invention also relates to a method for preserving a biological sample, characterized in that the biological sample is preserved in the presence of a polysaccharide or a salt thereof having a viscosity-average molecular weight of more than 3000 and not more than 500000, and containing a six-membered ring structure in its main chain, at -27°C or lower, and the preservation is such that, when the biological sample is preserved in the presence of the polysaccharide or the salt thereof for at least 5 months, the viability of the biological sample decreases by less than 5% based on the viability of the biological sample immediately before preservation. The polysaccharide salt is preferably a metal salt, a halogen salt, or a sulfate salt. The metal salt is preferably an alkali metal or alkaline earth metal salt. The alkali metal or alkaline earth metal may be sodium, potassium, calcium, or the like. The halogen may be chlorine, bromine, or the like.

[0038] The viscosity average molecular weight of the polysaccharide or its salt containing a six-membered ring structure in the main chain used in the method for preserving a biological sample of the present invention is desirably 400,000 or less, particularly 200,000 or less, because the viscosity can be adjusted to a low level and the solution is easy to handle as a cryopreservation solution.

[0039] A preferred method for preserving a biological sample is characterized in that the biological sample exhibits 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 polysaccharide or a salt thereof.

[0040] 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.

[0041] 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.

[0042] The method for preserving a biological sample, wherein the biological sample is a cell, is preferred.

[0043] A method for preserving a biological sample, wherein said biological sample is a mammalian cell, is preferred.

[0044] 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.

[0045] The present invention also relates to the use of a polysaccharide or its salt having a viscosity average molecular weight of more than 3000 and not more than 500000 and a six-membered ring structure in its main chain, for preserving a biological sample, preferably in vitro, at a temperature of -27°C or lower. The polysaccharide salt is preferably a metal salt, a halogen salt or a sulfate salt. The metal salt is preferably an alkali metal or alkaline earth metal salt. As the alkali metal or alkaline earth metal, sodium, potassium, calcium, etc. can be selected. As the halogen, chlorine, bromine, etc. can be used.

[0046] In the present invention, the viscosity average molecular weight of the polysaccharide or its salt containing a six-membered ring structure in the main chain 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 frozen storage solution.

[0047] 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.

[0048] In the present invention, the polysaccharide used for preserving a biological sample is preferably a polysaccharide other than dextran.

[0049] The present invention also relates to the use of a polysaccharide or a salt thereof having a viscosity-average molecular weight of more than 3,000 and not more than 500,000 and containing a six-membered ring structure in the main chain, for cryopreservation of a biological sample. The polysaccharide salt is preferably a metal salt, a halogen salt, or a sulfate salt. The metal salt is preferably an alkali metal or alkaline earth metal salt. As the alkali metal or alkaline earth metal, sodium, potassium, calcium, or the like can be selected. As the halogen, chlorine, bromine, or the like can be used.

[0050] In the present invention, the viscosity average molecular weight of the polysaccharide or its salt containing a six-membered ring structure in the main chain 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 frozen storage solution.

[0051] In the present invention, the polysaccharide used for cryopreserving a biological sample is preferably a polysaccharide other than dextran.

[0052] The present invention also relates to a cryopreservation agent for biological samples, comprising a polysaccharide or a salt thereof having a viscosity-average molecular weight of more than 3,000 and not more than 500,000 and a six-membered ring structure in its main chain. The polysaccharide salt is preferably a metal salt, a halogen salt, or a sulfate salt. The metal salt is preferably an alkali metal or alkaline earth metal salt. As the alkali metal or alkaline earth metal, sodium, potassium, calcium, or the like can be selected. As the halogen, chlorine, bromine, or the like can be used.

[0053] In the cryopreservation agent for biological samples of the present invention, the viscosity average molecular weight of the polysaccharide or its salt containing a six-membered ring structure in the main chain is desirably 400,000 or less, particularly 200,000 or less. This is because the viscosity can be adjusted low and the cryopreservation solution is easy to handle.

[0054] The polysaccharide as the main component of the cryopreservation agent is preferably a polysaccharide other than dextran.

[0055] A cryopreservation agent for biological samples that comprises a polysaccharide containing pentose, hexose or uronic acid or combinations thereof as repeating units is preferred.

[0056] A cryopreservation agent for biological samples that comprises a polysaccharide containing amino sugars as repeating units is preferred.

[0057] The functional groups of the polysaccharide or its salt used in the cryopreservation agent for biological samples of the present invention are preferably unmodified.

[0058] A cryopreservation agent for biological samples containing a polysaccharide or a salt thereof having a viscosity average molecular weight of 10,000 or more is preferred.

[0059] A cryopreservation agent for biological samples that includes a polysaccharide that is hyaluronic acid or pullulan is preferred.

[0060] 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.

[0061] In addition, because it does not contain serum and / 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. In addition, even cell-permeable and toxic compounds can be used at low concentrations that do not impair cell function.

[0062] The "viscosity average molecular weight" of the polysaccharide used in the present invention is calculated by the following method and formula:

[0063] 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 the polysaccharide 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 so that the relative viscosity of the stock solution to the standard solution is 2.0 to 2.4. (3) Dilute the 30°C stock solution 5 / 4, 5 / 3, and 5 / 2 times with the 30°C standard solution. (4) Measure the viscosity of the standard solution, undiluted solution, and diluted solution at 30°C. Use an E-type viscometer to measure the viscosity. (5) The viscosity of the original solution and 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: TIFF0007607703000001.tif2146Here, η sp : Reduced viscosity of polysaccharides [mL / g], η r : relative viscosity of polysaccharide [-], C: concentration of polysaccharide [g / mL]. (6) Plot the relationship between the polysaccharide concentration and the reduced viscosity of the polysaccharide, and draw an approximate line. The intercept of the approximate line (polysaccharide concentration = 0) is the limiting viscosity.

[0064] Viscosity average molecular weight: The viscosity average molecular weight is calculated from the intrinsic viscosity. TIFF0007607703000002.tif3559The viscosity average molecular weight M is calculated from the above Mark Hoying-Sakurada formula using the intrinsic viscosity derived by measurement and the values ​​of K and α published in literature, etc.

[0065] 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.

[0066] 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.

[0067] 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

[0068] The cryopreservation solution for biological samples of the present invention contains a polysaccharide or its salt having a viscosity average molecular weight of more than 3000 and not more than 500,000, and containing a six-membered ring structure in its main chain as a cryoprotectant, and can freeze the solvent portion into an amorphous glass state by cooling. This prevents the cell from bursting due to ice crystal formation, and also prevents damage to the cell and cell membrane because there is no volume expansion of water. If the viscosity average molecular weight of the polysaccharide is 3000 or less or exceeds 500,000, the survival rate of the biological sample after freezing is low. In addition, it is necessary for the polysaccharide to contain a six-membered ring structure in its main chain. This is because polysaccharides having a six-membered ring structure in their main chain are likely to form a polymer network structure around the cell through hydrogen bonds, and it is believed that the solvent portion can be vitrified even at a low concentration. For this reason, even if the concentration of the polysaccharide is low, a sufficient cryopreservation effect for the cells can be obtained. On the other hand, in the case of polysaccharides having a five-membered ring structure, such as fructan, it is difficult to stably form such a polymer network structure, and as a result, it is presumed that the concentration of the polysaccharide must be increased.

[0069] In the present invention, the viscosity average molecular weight of the polysaccharide or its salt containing a six-membered ring structure in the main chain 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 frozen storage solution.

[0070] In the present invention, it is preferable that the polysaccharide does not contain dextran.

[0071] Furthermore, since polysaccharides are biological components, they are lowly toxic. In addition, the cryopreservation solution for biological samples containing the polysaccharides of the present invention is a non-penetrating cryopreservation solution for intracellular samples, yet exhibits a high cryopreservation effect that could not be obtained by conventional techniques, and therefore can significantly reduce damage to and changes in the properties of the biological sample.

[0072] In addition, the cryopreservation solution for biological samples of the present invention does not contain intracellular penetrating chemicals such as DMSO and ethylene glycol, so it is considered that there is no risk of cytotoxicity or adverse effects on the properties of cells 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.

[0073] In addition, since it does not contain serum or serum-derived proteins, it is not contaminated with bacteria or viruses. However, it is possible to add proteins that are not contaminated with bacteria or viruses.

[0074] In addition, since the cryopreservation solution for biological samples of the present invention has a glass transition point at about -23°C ± 4, according to the method for freezing a biological sample, the method for freezing and preserving a biological sample, and the method for preserving a biological sample of the present invention, biological samples such as cells and tissues can be appropriately and 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 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 and ethylene glycol at low concentrations that do not impair the function of cells.

[0075] 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]

[0076] [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 frozen storage solution containing DMSO. [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 a cryopreservation solution containing various polysaccharides of the present invention on primary canine mesenchymal stem cells. [Figure 8] FIG. 1 shows the effect of adding various fragments. [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 shows the cell viability of primary canine mesenchymal stem cells cryopreserved using the saccharides of the present invention. [Figure 14A] FIG. 1 shows HPLC analysis results of test samples from various production examples. [Figure 14B]FIG. 1 shows the results of HPLC analysis of test samples from various production examples. [Figure 14C] FIG. 1 shows the results of HPLC analysis of test samples from various production examples. [Figure 14D] FIG. 1 shows HPLC analysis results for a calibration curve of saccharides having a given number of sugars. [Figure 15] FIG. 2 shows the results of HPLC analysis of a test sample having a polymer chain of the present invention. [Figure 16] FIG. 1 shows cell viability with test samples lacking polysaccharide structures. [Figure 17] FIG. 1 shows the cell viability when fructan is used as a test sample. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0077] The cryopreservation solution for biological samples of the present invention (hereinafter also referred to as the cryopreservation solution of the present invention) is a cryopreservation solution for biological samples that contains, in a solvent, a polysaccharide or a salt thereof that has a viscosity average molecular weight of more than 3,000 and not more than 500,000 and that contains a six-membered ring structure in its main chain.

[0078] In the present invention, the viscosity average molecular weight of the polysaccharide or its salt containing a six-membered ring structure in the main chain 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 frozen storage solution.

[0079] The polysaccharides are not particularly limited as long as they have a polysaccharide skeleton in the main chain and contain a six-membered ring structure in the main chain, and include those in which monosaccharides are bonded as repeating units through glycosidic bonds and their derivatives. For example, the monosaccharides include monosaccharides having 3 to 7 carbon atoms, or monosaccharides in which the hydroxyl group and / or hydroxymethyl group of the monosaccharide is substituted, such as monosaccharides in which the hydroxyl group and / or hydroxymethyl group is substituted with at least one substituent selected from the group consisting of a carboxyl group, an amino group, an N-acetylamino group, a sulfooxy group, a methoxycarbonyl group, and a carboxymethyl group. However, in the present invention, dextran is preferably excluded from the polysaccharides.

[0080] Examples of monosaccharides having 3 to 7 carbon atoms include triose, tetrose, pentose, hexose, and heptose, among which pentose or hexose is preferred. Examples of pentose include ribose, arabinose, xylose, lyxose, xylulose, ribulose, and deoxyribose. Examples of hexose include glucose, mannose, galactose, fructose, sorbose, tagatose, fucose, fuculose, and rhamnose.

[0081] Substituted monosaccharides include monosaccharides in which one or more hydroxymethyl groups of the monosaccharide are replaced with a carboxyl group, a methoxycarbonyl group or a carboxymethyl group, monosaccharides in which one or more hydroxyl groups of the monosaccharide are replaced with an amino group or an N-acetylamino group, monosaccharides in which one or more hydroxyl groups of the monosaccharide are replaced with a sulfooxy group, etc. The substituted monosaccharide may also be a substituted monosaccharide in which more than one of the hydroxymethyl and hydroxyl groups of the monosaccharide are replaced with a functional group as described above or a combination thereof.

[0082] For example, the monosaccharide substituted with a carboxyl group may be 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 may be 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 a plurality of substituents include N-acetylglucosamine-4-sulfate, iduronic acid-2-sulfate, glucuronic acid-2-sulfate, N-acetylgalactosamine-4-sulfate, neuraminic acid, and N-acetylneuraminic acid.

[0083] The polysaccharides used in the present invention are, for example, polymers containing the above-mentioned monosaccharides as repeating units. The chemical structure of the polysaccharides used in the present invention may be further partially modified or substituted as appropriate, to the extent that gelation does not occur. For example, the polysaccharides used in the present invention may be sulfated polysaccharides such as chondroitin sulfate in which one or more hydroxyl groups are substituted with sulfoxy groups. However, it is most desirable that the functional groups of the polysaccharides used in the present invention are not modified, that is, that no substituents are introduced into the sugar chains. The functional groups of the polysaccharides, particularly OH groups and NH 2 This is because hydrophilic groups such as the COOH group and the OH group are presumed to contribute to the protection of biological samples and the vitrification of the solvent, and therefore it is advantageous for improving the survival rate of biological samples if these functional groups are not modified.

[0084] The polysaccharide salt used in the present invention 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. As the alkali metal or alkaline earth metal, sodium, potassium, calcium or the like is selected. As the halogen, chlorine, bromine or the like can be used. It is believed that the polysaccharide salt lowers the freezing point of the solvent, thereby contributing to the vitrification of the solvent.

[0085] The polysaccharide used in the present invention can be, for example, a polysaccharide that contains pentose, hexose, or uronic acid or a combination thereof as a repeating unit.The polysaccharide can also be a polysaccharide that contains amino sugar as a repeating unit.For example, the polysaccharide can include hyaluronic acid, pullulan, chondroitin sulfate, methylcellulose, or modified sugar of hydroxyethyl starch.Preferably, the polysaccharide can be hyaluronic acid or pullulan.

[0086] In the present invention, it is preferable to use polysaccharides other than dextran as the polysaccharide.

[0087] The polysaccharide may be naturally derived or chemically synthesized. Commercially available polysaccharides may be used as they are. The monosaccharide forming the polysaccharide backbone may also be naturally derived, may be modified or substituted, or may be chemically synthesized. For example, the monosaccharide forming the polysaccharide backbone is preferably a biological component. However, the monosaccharide is not limited thereto. The monosaccharide forming the polysaccharide backbone preferably contains a hydrophilic group such as a hydroxyl group or a carboxyl group. In particular, the polysaccharide used in 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.

[0088] It is preferable that the cryopreservation solution containing the polysaccharide of the present invention does not contain dimethyl sulfoxide (DMSO). It is also preferable that the cryopreservation solution containing the polysaccharide of the present invention does not contain a compound having a cytotoxicity that is permeable to cells, such as ethylene glycol. In addition, in the cryopreservation solution containing the polysaccharide of the present invention, the molecular motion of the water in the solvent is restricted by the action of the polymeric sugar chain of the polysaccharide, so that the water can be solidified and / or frozen in a vitrified state without crystallization. The freezing method called the normal vitrification method is a method applied to cells with a particularly low survival rate after thawing, in which the solute (a cryoprotectant for preventing freezing damage) is excluded from the crystals when the solution is frozen, thereby 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. Therefore, in such a method, the concentration of the solute (cryoprotectant) is increased and the cooling rate is increased to make it easier to vitrify water. However, it has been known that the problem of increasing the osmotic pressure difference also increases damage to cells, and that recrystallization occurs during lysis, causing damage to the cells. In addition, there are difficulties in terms of technique.

[0089] In addition, the cryopreservation solution containing the polysaccharide of the present invention does not contain serum or serum-derived proteins, and therefore is not contaminated with bacteria or viruses, although it is possible to add proteins that are not contaminated with bacteria or viruses.

[0090] According to the cryopreservation solution containing the polysaccharide or its salt of the present invention, solvent molecules can be trapped in the matrix formed by the polymer chains during the cooling process. This causes the inside of the cells to be dehydrated and vitrified, suppressing the formation of ice crystals in the cells, and further weakening the osmotic shock in the cells during freezing, which is a problem in conventional vitrification methods. Since the cryoprotectant is a polysaccharide, it is considered that the damage to cells caused by the cryopreservation solution of the present invention is low, in addition to the low cytotoxicity in the first place, since there is no need to increase the concentration of solutes to restrict the molecular motion of water. Furthermore, 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, according to the cryopreservation solution of the present invention, biological samples can be efficiently frozen and preserved by a simple method with reduced toxicity.

[0091] The polysaccharide or its salt as the cryopreservation agent contained in the cryopreservation solution of the present invention preferably has a viscosity average molecular weight of more than 3000 and not more than about 500000. In the present invention, the viscosity average molecular weight of the polysaccharide or its salt containing a six-membered ring structure in the main chain is preferably not more than 400000, particularly not more than 200000. This is because the viscosity can be adjusted low and the cryopreservation solution is easy to handle.

[0092] The "viscosity average molecular weight" of the polysaccharide 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) Dissolve the polysaccharide sample in a standard solution at 30°C to prepare a stock solution. If the polysaccharide sample is available in solution, the solids obtained by removing the solvent from the solution are used as the polysaccharide sample. In the case of a mixed sample containing multiple polysaccharides, each polysaccharide is separated and fractionated, and the solvent is removed from each substance to obtain a sample of each water-soluble polymer. If the polysaccharide is unknown, the substance is identified by HPLC, LC-MS, LC-IR, etc. In addition, if multiple unknown polysaccharides are included, each component is separated and fractionated, and the substance of each polysaccharide is identified by HPLC, LC-MS, LC-IR, etc., and the viscosity average molecular weight is calculated as described below. Even if the mixture contains impurities in the polysaccharide, if they do not affect the viscosity (for example, impurities such as metal salts), the mixture is used as the polysaccharide sample. In addition, if the mixture contains impurities that affect the calculation of the viscosity average molecular weight, the impurities are removed or the polysaccharides are fractionated before measurement. At this time, the viscosity of each of the standard solution and the stock solution is measured, and the relative viscosity of the stock solution to the standard solution is adjusted to be 2.0 to 2.4. (3) Dilute the 30°C stock solution 5 / 4, 5 / 3, and 5 / 2 times with the 30°C standard solution. (4) Measure the viscosity of the standard solution, undiluted solution, and diluted solution at 30°C. Use an E-type viscometer to measure the viscosity. (5) The viscosity of the original solution and 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: TIFF0007607703000003.tif2146Here, η sp : Reduced viscosity of polysaccharides [mL / g], η r : relative viscosity of polysaccharide [-], C: polysaccharide concentration [g / mL]. (6) Plot the relationship between the polysaccharide concentration and the reduced viscosity of the polysaccharide, and draw an approximate straight line. The intercept of the approximate straight line (where the polysaccharide concentration is 0) is taken as the limiting viscosity. Viscosity average molecular weight: The viscosity average molecular weight is calculated from the intrinsic viscosity. TIFF0007607703000004.tif3559The viscosity average molecular weight M is calculated from the intrinsic viscosity obtained by measurement and the K and α values ​​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 for fructans, K = 1.6 × 10 -3 , α=0.45 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] As described above, if the polysaccharide to be used is unknown, the polysaccharide is identified by HPLC, LC-MS, LC-IR, or the like, and then K and α are determined.

[0096] In the present invention, the molecular weight calculated by this method is defined as the viscosity average molecular weight.

[0097] By setting the viscosity-average molecular weight of the polysaccharide or its salt within the above range, when the cryopreservation solution of the present invention containing the polysaccharide or its salt in a solvent is cooled, the amorphous glass state in the frozen state is stabilized. Since cooling and freezing do not cause damage to cells, cells are stably and efficiently cryopreserved. Therefore, the survival rate of cells in the biological sample after thawing the biological sample after cryopreservation is high. If the viscosity-average molecular weight of the polysaccharide is 3000 or less, vitrification is unlikely to occur well. In addition, if the viscosity-average molecular weight of the polysaccharide is greater than 500,000, the viscosity increases significantly, and the solubility decreases, or the prepared solution foams, which may cause problems in terms of poor handling. Therefore, when using a naturally derived polymeric polysaccharide or a commercially available polymeric sugar chain, it is preferable to adjust the molecular weight by performing a treatment such as hydrolysis, enzyme treatment, or subcritical treatment. The viscosity-average molecular weight is particularly preferably 10,000 or more. Moreover, polysaccharides having a viscosity average molecular weight of 400,000 or less are preferred, more preferably 200,000 or less, and particularly preferably 150,000 or less, because the viscosity can be adjusted low and they are easy to handle as a cryopreservation solution.

[0098] The cryopreservation solution of the present invention uses a polysaccharide or its salt having a viscosity average molecular weight of more than 3000 and about 500,000 or less, and containing a six-membered ring structure in its main chain, and thus exhibits a high cell protection effect without the need to add protein components such as serum derived from humans or cows or serum-derived components (e.g., albumin, etc.). Therefore, it is considered that there is no need to worry about infectious diseases, and that there is no influence of differences between lots of biological preparations. It is possible to add proteins that do not cause infectious diseases. In addition, since the cryopreservation solution of the present invention has low cytotoxicity, cells after thawing have a high survival rate. 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 thawing. In other words, it is considered that the glass state of the biological sample in the frozen state is stabilized by cryopreservation using the polysaccharide of the present invention.

[0099] The viscosity average molecular weight of the polysaccharide or salt thereof containing a six-membered ring structure in the main chain is desirably 400,000 or less, particularly 200,000 or less, because the viscosity can be adjusted to a low level and the solution is easy to handle as a cryopreservation solution.

[0100] 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.

[0101] 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 agent of the cryopreservation solution of the present invention is a polysaccharide, so it is considered not to change the properties of cells. Therefore, it is considered that biological samples can be cryopreserved while maintaining the properties of cells.

[0102] The cryopreservation solution of the present invention contains the polysaccharide or its salt of the present invention 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 polysaccharide or its salt is preferably 5 w / v% or more, and particularly preferably 10 w / v% or more. Also, the concentration of the polysaccharide or its salt is preferably 50 w / v% or less, and particularly preferably 20 w / v% or less. Preferably, the concentration of the polysaccharide or its salt is 5 w / v% or more and 20 w / v% or less.

[0103] The cryopreservation solution of the present invention may further contain a cell-protecting component. Examples of such cell-protecting components include compounds having a viscosity-average molecular weight of more than 3000 and less than that of polysaccharides having a viscosity-average molecular weight of 500,000 or less. When such a compound is present in the cryopreservation solution, the water molecules near the cell membrane are replaced by the cell-protecting component, suppressing the formation and growth of ice crystals near the cell membrane, and as a result, cell membrane damage can be further suppressed.

[0104] Specifically, for example, the cell protective component is a saccharide that is a component of the polysaccharide of the present invention or a fragment of the polysaccharide of the present invention. For example, monosaccharides, disaccharides, or oligosaccharides having a viscosity average molecular weight of less than 3000, preferably 2000 or less, and more preferably 1000 or less, are exemplified. In the case of monosaccharides, disaccharides, and compounds 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 the viscosity average molecular weight.

[0105] The cell-protecting component that can be used in the present invention is not particularly limited as long as it does not impair the effects of the present invention, but particularly preferred examples include polysaccharides, such as cleavage products (fragments) of glycosaminoglycans, etc. The cleavage products of glycosaminoglycans can be monosaccharides, disaccharides, or oligosaccharides that constitute glycosaminoglycans.

[0106] Preferably, the cleavage product of glycosaminoglycan is the cleavage product of hyaluronic acid.Therefore, preferably, the cleavage product of glycosaminoglycan is glucuronic acid or N-acetylglucosamine, or a disaccharide or oligosaccharide thereof.More preferably, it can be glucuronic acid, or a disaccharide or oligosaccharide thereof.

[0107] The "cleavage product" of the present invention means a compound having a viscosity average molecular weight of more than 3000 and smaller than that of the polysaccharides used in the present invention having a viscosity average molecular weight of 500,000 or less, which is considered to be obtained when polysaccharides are subjected to hydrolysis, enzyme treatment, subcritical treatment, etc. Therefore, the cleavage product may be a monosaccharide and / or a polymer of monosaccharides having various degrees of polymerization, which are components of the corresponding polysaccharide, and / or a mixture thereof. In this specification, "subcritical treatment" means contacting a subcritical fluid as an extraction solvent that has been made subcritical under conditions of a predetermined temperature and a predetermined pressure with the raw material to be extracted, i.e., a high molecular weight polysaccharide. 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 near the critical point is called subcritical water. Using the hydrolysis action of this subcritical water, a desired component can be obtained from the raw material to be extracted. The conditions for the subcritical treatment of polymeric polysaccharides are, for example, a temperature of 150°C or higher and 350°C or lower, and the subcritical treatment pressure can be equal to or higher than the saturated vapor pressure at each temperature, for example, 0.5 MPa or higher and 25 MPa or lower. After the subcritical treatment, components having a molecular weight of a given or lower can be separated and collected and used as the cleavage product in the present invention. There are no particular limitations on the hydrolysis or enzyme treatment, and reagents and treatment methods that are commonly used can be used without problems.

[0108] The cytoprotective component used in the present invention may be contained in the cryopreservation solution at a concentration of about 1 w / v% or more and 10 w / v% or less. That is, when a cytoprotective component is additionally used, the ratio of polysaccharide to cytoprotective component is preferably about 10:1. If the concentration of the cytoprotective component is less than 1 w / v%, the cytoprotective effect of the cytoprotective component may not be sufficiently obtained. Moreover, even if the cytoprotective component is added to a concentration of 10 w / v% or more, it is difficult to obtain further effects as a cytoprotective component.

[0109] 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 a cell-permeable and cytotoxic chemical substance such as DMSO that can act as a differentiation factor, 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.

[0110] 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.

[0111] 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-sized 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.

[0112] 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.

[0113] In addition, by using polysaccharides that are biological components as the polysaccharides used in the present invention, it is possible to thaw frozen biological samples and use them as a solution for cell administration.Furthermore, in the case of tissue or tissue-like material, polysaccharides can be added at the organization stage and frozen and stored as is.This is thought to reduce problems after transplantation.In such a case, the preferred polysaccharide 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.

[0114] 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.

[0115] The cryopreservation method of the present invention includes the steps of: placing a biological sample in a cryopreservation solution containing a polysaccharide or a salt thereof having a viscosity average molecular weight of more than 3000 and not more than 500,000 and containing a six-membered ring structure in the main chain in a solvent; freezing the cryopreservation solution containing the biological sample; and preserving the biological sample by holding the cryopreservation solution containing the biological sample at a temperature of -27°C or lower. The salt of the polysaccharide may be a metal salt, a halogen salt, or a sulfate salt of the polysaccharide. As the metal salt, a salt of an alkali metal or an alkaline earth metal is preferable. As the alkali metal or the alkaline earth metal, sodium, potassium, calcium, or the like is selected. As the halogen, chlorine, bromine, or the like can be used. Since the cryopreservation solution containing the biological sample is frozen 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, and then stored as is, the cryopreservation can be performed, and therefore, there is no need for a rapid operation for rapid cooling required in the conventional vitrification method. It is therefore considered that the operability can be improved and a stable preservation effect can be obtained at the same time.

[0116] The viscosity average molecular weight of the polysaccharide or its salt containing a six-membered ring structure in the main chain is desirably 400,000 or less, particularly 200,000 or less, because the viscosity can be adjusted to a low level and the solution is easy to handle as a cryopreservation solution.

[0117] 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.

[0118] 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.

[0119] In addition, the cryopreservation solution of the present invention may be pH adjusted as necessary. For example, when a polysaccharide having a carboxylic acid group or the like is used as the polysaccharide of the present invention, the cryopreservation solution may be acidic. 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 cryopreserved cells, 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.

[0120] 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

[0121] 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".

[0122] 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 performed at a processing temperature of 175°C, a processing pressure of 0.89MPa, and a processing time of 3 minutes. The subcritical processing product was then dried by freeze-drying or spray-drying. As a result, a test sample of hyaluronic acid with a viscosity average molecular weight of about 10,000, that is, the cryopreservation agent of the present invention, was obtained. The intrinsic viscosity was 0.49dL / g.

[0123] 1 g of this test cryopreservation agent was dissolved in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, 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, manufacturing 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] Example 2 Hyaluronic acid with a viscosity average molecular weight of 50,000 (Lifecore Biomedical; powder) was used as the test sample for the cryopreservation agent of the present invention. 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 Example 2 (i.e., the concentration of the test sample hyaluronic acid in the test cryopreservation solution is 10 w / v%). The intrinsic viscosity was 1.67 dL / g.

[0128] Example 3 Hyaluronic acid with viscosity average molecular weight of 15000 (lifecore biomedical; powder) 1g is dissolved in αMEM medium (Gibco, product number C1257-1500BT, solvent is water) 10mL to obtain a test sample solution with hyaluronic acid concentration of 10w / v%, and the test sample (hyaluronic acid fragment sample with viscosity average molecular weight of 1000) of Preparation Example 1 is added to the test sample solution with a final concentration of 1w / v% to obtain the test cryopreservation solution of Example 3. The intrinsic viscosity is 0.65dL / g.

[0129] Example 4 The pH of the test cryopreservation solution of Example 3 (containing hyaluronic acid (manufactured by Lifecore Biomedical) with a viscosity average molecular weight of 15000 and the test sample of Production Example 1) was adjusted to neutral using 10 mM Tris-HCl to obtain the test cryopreservation solution of Example 4. The intrinsic viscosity was 0.65 dL / g.

[0130] Example 5 A test cryopreservation solution of Example 5 was obtained by dissolving 1 g of pullulan (Tokyo Chemical Industry Co., Ltd.) with a viscosity average molecular weight of 373,000 in 10 mL of α-MEM medium (Gibco, product number C1257-1500BT, the solvent is water) (i.e., the concentration of pullulan, the test sample, in the test cryopreservation solution was 10 w / v%). The intrinsic viscosity was 0.55 dL / g.

[0131] Example 6 The test sample (hyaluronic acid fragment sample with viscosity average molecular weight of 1000) of Preparation Example 1 was added to the test cryopreservation solution (containing pullulan with viscosity average molecular weight of 373000) of Example 5 at a final concentration of 1 w / v% to obtain the test cryopreservation solution of Example 6. The intrinsic viscosity was 0.55 dL / g.

[0132] 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.

[0133] Example 8 The test sample (hyaluronic acid fragment sample with viscosity average molecular weight of 1000) of Preparation Example 1 is added to the test cryopreservation solution of Example 5 (containing pullulan with viscosity average molecular weight of 373000) at a final concentration of 5w / v% to obtain a test cryopreservation solution.The intrinsic viscosity is 0.55dL / g.

[0134] Example 9 The pH of the test cryopreservation solution of Example 8 (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 a test cryopreservation solution of Example 9. The intrinsic viscosity was 0.55 dL / g.

[0135] Example 10 Hyaluronic acid with a viscosity average molecular weight of 15000 (Lifecore Biomedical; powder) was dissolved in 10 mL of αMEM medium (Gibco, product number C1257-1500BT, solvent is water) to obtain a test sample solution with a hyaluronic acid concentration of 10 w / v%, and sucrose (Nacalai Tesque, Inc.) was added to the test sample solution at a final concentration of 1 w / v% to obtain a test cryopreservation solution of Example 10. The intrinsic viscosity was 0.65 dL / g.

[0136] Example 11 1g of hyaluronic acid (lifecore biomedical; powder) with viscosity average molecular weight of 15000 is dissolved in 10mL of αMEM medium (Gibco, product number C1257-1500BT, solvent is water) to obtain a test sample solution with a hyaluronic acid concentration of 10w / v%, and glucuronic acid (FUJIFILM Wako Pure Chemical Industries, Ltd.) is added to the test sample solution with a final concentration of 1w / v% to obtain the test frozen storage solution of Example 11. The intrinsic viscosity is 0.65dL / g.

[0137] 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%).

[0138] 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.

[0139] 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.

[0140] Comparative Example 4 1g of high molecular weight hyaluronic acid (manufactured by Shanghai Easier Industrial Development Co., Ltd.) with a viscosity average molecular weight of 1,000,000 was dissolved in 100mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, solvent is water) as a solvent to obtain a cryopreservation solution for testing of Comparative Example 4 (i.e., the concentration of hyaluronic acid, which is the test sample in the cryopreservation solution for testing, is 1w / v%). Note that, the cryopreservation solution for testing of Comparative Example 4 was prepared so that the hyaluronic acid concentration was 1w / v%, since the concentration of 10w / v% in the medium could not be obtained due to handling problems. The limiting viscosity was 17.2dL / g.

[0141] Experimental Example 1 A test sample solution of Experimental Example 1 was obtained by dissolving 1 g of dextran (MP Co., Ltd.; powder) having a viscosity average molecular weight of 42,500 in 10 mL of α-MEM medium as a solvent (i.e., the concentration of the dextran in the test sample solution was 10 w / v%). The intrinsic viscosity was 0.19 dL / g.

[0142] Comparative Example 6 Fructan derived from shallots is obtained according to Example 2 of JP 2012-235728 A.

[0143] Approximately 1 kg of dried shallots as raw material was crushed and mixed with water for extraction in which 15 g of calcium hydroxide had been suspended, and extraction was carried out at room temperature for 1 hour, followed by heating extraction at 95°C for 1 hour, and then heating extraction overnight at 60°C. Activated carbon was added to the extract for deodorization, and then it was filtered through diatomaceous earth to remove the residue and activated carbon.

[0144] The obtained extract was passed through an ultrafiltration membrane to purify the fructan.

[0145] The purified liquid was once heat-sterilized, and then decolorized again by adding activated carbon. The purified liquid was sterilized by heating at 85°C for 1 hour, freeze-dried, and powdered to obtain 400 g of purified fructan powder.

[0146] The viscosity average molecular weight of the resulting purified fructan powder was measured and found to be 12000. The intrinsic viscosity was 0.11 dL / g.

[0147] 1 g of this fructan powder was dissolved in 10 mL of α-MEM medium as a solvent to obtain a test sample solution of Comparative Example 6 (i.e., the test sample fructan concentration in the test sample solution was 10 w / v %).

[0148] <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 Examples 1 and 2 and Comparative Examples 1, 2 and 4 at a concentration of 1000 cells / mL.

[0149] 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.

[0150] 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 Examples 1 and 2 and Comparative Examples 1 and 4. From FIG. 1, it can be seen that a good preservation effect was obtained in the test cryopreservation solutions of Examples 1 and 2. In particular, in the case of the test cryopreservation solution of Example 1, in which a hyaluronic acid sample with a viscosity average molecular weight of about 10,000 obtained by subcritical processing of hyaluronic acid is used as the cryopreservation agent in the cryopreservation solution, a high preservation effect exceeding 90% was obtained. This preservation effect was even higher than that of Comparative Example 1, which contains DMSO, a commonly used cryopreservation agent, as the cryopreservation agent. It can be seen that the cryopreservation solution of the present invention is an excellent cryopreservation solution with low cytotoxicity. In addition, in Comparative Example 4, which contains a high molecular weight hyaluronic acid with a viscosity average molecular weight of 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%. From these results, it was confirmed that polysaccharides having a viscosity average molecular weight of about 200,000 or less are effective as cryoprotectants. In addition, since the cryopreservation agent of the present invention exhibits high cell preservation effects 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 a cell culture solution to suspend the cells, and then frozen and preserved. There is no need to centrifuge the cells to be preserved, and it is believed that a higher cell survival rate and maintenance of cell properties are possible.

[0151] <Test Example 2: Evaluation of cryopreservation solution analysis by differential scanning calorimetry> The test samples of Examples 1 and 2 and Comparative Example 2 were prepared to a final concentration of 10% by using ultrapure water instead of α-MEM 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.

[0152] 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 Example 2, in which the test sample was hyaluronic acid with a viscosity average molecular weight of 50,000, 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). In Example 1, the ice crystal melting peak was extremely small and almost not observed, and further, a glass transition was confirmed near -23°C ± 4°C as shown in Figure 2B.

[0153] 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.

[0154] The results are shown in FIG. 3A (Comparative Example 2), FIG. 3B (Comparative Example 1), and FIG. 3C (Example 1), respectively.

[0155] In Comparative Example 2, in which the test sample was ultrapure water, a very large heat generation peak associated with ice crystal formation was observed during the temperature drop process, as shown in FIG. 3A. Also, a large heat of fusion associated with ice crystal melting was observed during the temperature rise process. In Comparative Example 1, in which the test sample was 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 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.

[0156] <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.

[0157] 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%.

[0158] 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.

[0159] 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 was nearly 100% of the cell viability immediately before storage in the cryopreservation solution of the present invention, 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.

[0160] <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 6 The 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.

[0161] 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.

[0162] 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.

[0163] 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 a remarkable effect of being capable of cryopreserving a biological sample while maintaining its properties without exposing the biological sample to stress, unlike conventional cryopreservation solutions. It is possible to add cytotoxic chemical substances such as DMSO and ethylene glycol at low concentrations that do not impair the function of the cells.

[0164] 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.

[0165] <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 × 106 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.

[0166] 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.

[0167] 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.

[0168] <Test Example 6: Evaluation of cryopreservation solutions containing various polysaccharides> Cultured primary canine mesenchymal stem cells (cyagen C160) were added at 1 × 10 6The cells were suspended in the test cryopreservation solutions (serum-free) of Examples 1, 3 to 9 and Experimental Example 1 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.

[0169] From FIG. 7, it can be seen that polysaccharides having a predetermined molecular weight, including not only hyaluronic acid but also other polysaccharides such as pullulan, show good cell protection effects as cryoprotectants in cryopreservation solutions, regardless of the type of polysaccharide. Dextran showed only low cell protection effects as a cryopreservation agent. Furthermore, the cell preservation effect was further improved by using a combination of polysaccharide and fragments, which are the cleavage products of polysaccharides. When fragments are added, the prepared cryopreservation solution may be acidic. In such cases, a higher cell protection effect was obtained by neutralizing the cryopreservation solution by adjusting the pH. It is believed that the pH adjustment provides more suitable conditions for cell survival. Furthermore, when fragments are added, it was preferable to add them in an amount of 1 to 5 w / v% to the test sample, which is a polysaccharide, in order to further improve the cell protection effect. Even if the amount of fragments added was more than 10 w / v% to the test sample, no further effect on the improvement of cell viability was observed.

[0170] 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 but also on canine mesenchymal stem cells.

[0171] <Test Example 7: Evaluation of the effect of adding various fragments> Cultured primary canine mesenchymal stem cells (cyagen C160) were added at 1 × 10 6The cells were suspended in the test cryopreservation solutions (serum-free) of Examples 10 and 11 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.

[0172] As shown in Fig. 8, a higher cell protection effect was observed when glucuronic acid and the hyaluronic acid fragment sample of Production Example 1 obtained by subcritical treatment and having a viscosity average molecular weight of 1000 were added, compared to sucrose, which is sometimes added to conventional cryopreservation solutions and is considered to be preferable from the viewpoint of water retention. From this result, it is considered that as additional additives, fragments that are the cleavage products of glycosaminoglycans, particularly fragments that are the cleavage products of hyaluronic acid and glucuronic acid, which is a constituent monosaccharide of hyaluronic acid, are preferable.

[0173] <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.

[0174] 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.

[0175] This result indicates that the cryopreservation solution of the present invention freezes the cells in a vitrified state.

[0176] <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 1000 / 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 α-MEM medium without a cryopreservative. Then, 5 μL of the cell suspension and the control suspension 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. 10A and 10B.

[0177] 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.

[0178] 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).

[0179] 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.

[0180] <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.

[0181] 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.

[0182] <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 ...

[0183] 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.

[0184] <Test Example 12: Evaluation of cryoprotective effect during cryopreservation using sugars with low molecular weight> Cultured primary canine mesenchymal stem cells (cyagen C160) were added at 1 × 10 6 Each test sample of Production Examples 1 to 3 was suspended in each test cryopreservation solution prepared by dissolving each test sample in αMEM medium to a concentration of 10 w / v% at a concentration of 10 cells / mL. The cell suspension containing each test cryopreservation solution was then frozen in a slow cell freezer (Nalgene (registered trademark) Mr. Frosty) in a -80°C freezer. After 7 days of cryopreservation, the cell suspension containing each test cryopreservation solution was removed and rapidly thawed in a 37°C warm bath. The cell viability of the cell suspension containing each test cryopreservation solution after thawing was evaluated by trypan blue staining. The results are shown in FIG. 13.

[0185] As shown in Figure 13, saccharides having a molecular weight smaller than that of polymers alone cannot provide a cell survival effect. 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.

[0186] <Test Example 13: HPLC analysis of subcritically treated hyaluronic acid samples> A 1 wt% aqueous solution of the test samples of Production Examples 1 to 3 was prepared and filtered through a 0.45 μm membrane filter (Millipore), after which the components of each test sample were analyzed by HPLC. 2 PO 4 Aqueous solution, B solution: 800mM NaH 2 PO 4 Using an aqueous solution, ZORBAX NH 2The components were separated using a normal phase HPLC column (Agilent Technologies, column size φ4.6×250 mm, particle size 5 μm) at a flow rate of 1.0 mL / min, column temperature of 40°C, and 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 14A-C. In addition, as standards, aqueous solutions containing hyaluronic acid oligosaccharides HA02 (disaccharide), HA04 (tetrasaccharide), HA06 (hexasaccharide), HA08 (octasaccharide), and HA10 (decasaccharide) (all manufactured by Iduron) at a concentration of 0.2 wt% were prepared and subjected to HPLC analysis in the same manner. The results are shown in Figure 14D.

[0187] 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 14D. Figure 14A 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.

[0188] <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.

[0189] Similarly, when compared with Fig. 14D, 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.

[0190] <Test Example 15: Evaluation of cell viability using a test sample without a polysaccharide structure> Instead of the polysaccharide of the present invention, a test cryopreservation solution containing gelatin of Comparative Example 3 was used to evaluate the cell viability after cryopreservation. 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 ...

[0191] 16, when gelatin was used as the cryopreservation agent, a significantly lower cell viability was obtained compared to Comparative Example 1, in which DMSO was used as the cryopreservation agent. Therefore, it can be seen that the cryopreservation solution of the present invention exhibits a high cell viability effect by containing the polysaccharide of the present invention.

[0192] <Test Example 16: Comparative evaluation with fructan> Under the same conditions as in Test Example 1, the cell viability was measured in the case of using the test cryopreservation solution containing hyaluronic acid with a viscosity average molecular weight of about 10,000 as a cryopreservation agent in Example 1, and in the case of using the test cryopreservation solution containing fructan with a viscosity average molecular weight of 12,000 as a cryopreservation agent in Comparative Example 6. When using the cryopreservation solution in Example 1, the cell viability was 92%, while it was 40% in the cryopreservation solution in Comparative Example 6.

[0193] When comparing at the same concentration (10w / v%) as a cryopreservation agent in a cryopreservation solution, it is found that hyaluronic acid with a six-membered ring structure has a better cell preservation effect than fructan. Figure 17 shows a graph of Table 5 in JP 2012-235728 A (concentration on the horizontal axis is w / v%), and shows that the cell survival rate is high when the concentration of fructan is in the range of 20% to 60%, and in the case of fructan with a five-membered ring structure, it cannot be said that it functions sufficiently as a cryopreservation solution unless the concentration is high.

[0194] From the above results, it can be seen that the cryopreservation solution for biological samples containing the polysaccharide 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, without basically requiring the addition of cell-permeable and cytotoxic compounds such as DMSO and ethylene glycol, and / or serum or serum-derived proteins. The cells are well protected and their properties are maintained. Furthermore, when the cryopreservation solution for biological samples of the present invention further contains a polysaccharide fragment component, the vitrification state of the cells is further stabilized, and a higher cell viability can be obtained.

Claims

1. A cryopreservation solution for biological samples, comprising pullulan or a salt thereof (excluding cross-linked pullulan) having a viscosity average molecular weight of more than 3,000 and not more than 500,000 dissolved in an aqueous solvent.

2. The cryopreservation solution according to claim 1 , wherein the pullulan has no functional groups modified.

3. 3. The cryopreservation solution according to claim 1, wherein the pullulan has a viscosity average molecular weight of 10,000 or more.

4. The cryopreservation solution according to any one of claims 1 to 3, wherein the concentration of pullulan in the cryopreservation solution is 1 w / v % or more and 50 w / v % or less.

5. The cryopreservation solution according to any one of claims 1 to 4, which is an intracellular non-permeable cryopreservation solution.

6. The cryopreservation solution according to any one of claims 1 to 5, wherein the biological sample is a cell, a tissue, or a tissue-like substance which is a membrane or an aggregate.

7. The cryopreservation solution according to claim 6, wherein the biological sample is a mesenchymal stem cell, a blood cell, an endothelial cell, or a tissue for transplantation.

8. The cryopreservation solution according to claim 6, wherein the biological sample is a sperm, an egg, or a fertilized egg.

9. 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 8.

10. A step of placing a biological sample in a cryopreservation solution in which pullulan or a salt thereof (excluding crosslinked pullulan) having a viscosity average molecular weight of more than 3,000 and not more than 500,000 is dissolved in an aqueous solvent; 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:

11. The method for cryopreserving a biological sample according to claim 10, wherein the step of immersing the biological sample in the cryopreservation solution is carried out before cooling the biological sample.

12. 12. The method for freezing and storing a biological sample according to claim 10 or 11, wherein the biological sample is a cell, a tissue, or a tissue-like substance which is a membrane or an aggregate.

13. The method for freezing and preserving a biological sample according to claim 12, wherein the biological sample is a sperm, an egg, or a fertilized egg.

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