Aqueous polymer solution for cryopreservation and solidified body containing biological samples

Abstract

To provide an aqueous polymer solution for the cryopreservation of a biological sample and a solidified body containing a biological sample, with high cell survival rate.SOLUTION: Provided are an aqueous polymer solution containing a water-soluble polymer or a salt thereof in an aqueous solvent and used for the cryopreservation of a biological sample, the intrinsic viscosity (η) of the aqueous polymer solution being 0.20 dL / g or more and 0.95 dL / g or less, the orthogonal projected area of the biological sample when solidified at -80°C having decreased to less than 8 / 10 compared to before solidification, and in the differential scanning calorimeter (DSC) curve of a temperature cooling process using a DSC, the calorific value of an exothermic peak during the temperature cooling process being 0 J / g, or 65% or less of the corresponding calorific value of the standard solution consisting of water; and a frozen solidified body thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an aqueous polymer solution for cryopreservation of a biological sample and a solidified body containing the biological sample. [Background technology]

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

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

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

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

[0006] Patent Document 3 describes a cryopreservation composition containing a carboxylated polyamino acid in which the amino group of the polyamino acid is blocked by carboxylation (or acetylation) with a carboxylic anhydride and an organic amphoteric agent. Patent Document 4 further discloses fructan as an active ingredient of a cell preservation solution. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Publication No. 63-216476 [Patent Document 2] International Publication No. 2016 / 076317 [Patent Document 3] Special Publication No. 2018-533377 [Patent Document 4] JP 2012-235728 A [Non-patent literature]

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

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

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

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

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

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

[0014] Furthermore, the fructan described in Patent Document 4 also caused cell death due to rupture or other reasons, and was not capable of adequately cryopreserving cells.

[0015] 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.

[0016] The present invention has been made in view of the above problems, and provides a polymer aqueous solution for cryopreservation to appropriately preserve biological samples. In particular, the present invention aims to provide a polymer aqueous solution for cryopreservation that enables stable long-term storage of biological samples at temperatures below -27°C without essentially using chemical substances such as dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), etc., and without essentially adding serum or serum-derived proteins, a method for preventing or suppressing the growth of ice crystals in a biological sample using the same, and a solidified body containing a biological sample. [Means for solving the problem]

[0017] The present invention relates to an aqueous polymer solution containing a water-soluble polymer or a salt thereof in an aqueous solvent and used for cryopreservation of a biological sample, the aqueous polymer solution having an intrinsic viscosity (η) of 0.20 dL / g or more and 0.95 dL / g or less, the orthogonal projected area of ​​the biological sample in a state where the aqueous polymer solution is solidified at -80°C is reduced to less than 8 / 10 of the orthogonal projected area of ​​the biological sample in a culture solution (in a culture medium) before solidification, and a differential scanning calorimeter (DSC) curve of the aqueous polymer solution during a temperature drop obtained under the measurement conditions (1) to (2) below shows that the heat generation amount of an exothermic peak during the temperature drop is 0 J / g or 65% or less of the corresponding heat generation amount of a reference solution consisting of water. Here, the DSC scan was performed under the following conditions: (1) holding at 20°C for 1 minute, followed by cooling to -80°C at a rate of 5°C / min, and (2) holding at -80°C for 1 minute, followed by heating to 20°C at a rate of 10°C / min.

[0018] In the aqueous polymer solution of the present invention, it is preferable that in a DSC curve during a temperature rise obtained using a differential scanning calorimeter (DSC) under the measurement conditions (1) to (2) above, no endothermic peak is observed as measured by DSC, or the temperature at which an endothermic peak is observed is higher than -1.4°C and 1.1°C or lower.

[0019] In addition, it is desirable that the orthogonal projection area of ​​the biological sample in the state where the polymer aqueous solution is solidified at -80°C is at least 1 / 3.5 of the orthogonal projection area of ​​the biological sample in the culture solution (culture medium) before solidification.

[0020] The intrinsic viscosity (η) of the aqueous polymer solution is preferably 0.32 dL / g or more and 0.65 dL / g or less.

[0021] It is also preferable that the temperature at which the endothermic peak is observed is -1.3°C or higher and 0.83°C or lower.

[0022] In this specification, "no endothermic peak is observed" means that no endothermic peak is observed in the range of -30°C to +5°C. If water containing a water-soluble polymer shows an endothermic peak, it is generally in this range. In addition, the orthogonal projection area of ​​the biological sample and the Munsell color brightness of the region occupied by the biological sample and the matrix region are temperature-independent and invariant after freezing and solidifying. However, if the orthogonal projection area of ​​the biological sample and the Munsell color brightness of the region occupied by the biological sample and the matrix region are temperature-dependent, the orthogonal projection area and the Munsell color brightness are measured at -80°C.

[0023] The salt of the water-soluble polymer is preferably, for example, a metal salt, a halogen salt, or a sulfate salt. The metal salt is preferably an alkali metal or an 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.

[0024] The aqueous polymer solution for cryopreservation of biological samples of the present invention preferably does not contain dimethyl sulfoxide, which is an intracellular permeable cryoprotectant. Also, the aqueous polymer solution for cryopreservation of biological samples of the present invention preferably does not contain cytotoxic cryoprotectants such as ethylene glycol, because these are harmful to cells after thawing.

[0025] In the aqueous polymer solution of the present invention, in a DSC curve during a temperature drop obtained under the above-mentioned DSC measurement conditions (1) to (2), the calorific value of an exothermic peak during the temperature drop is preferably 0 J / g or 40% or less of the corresponding calorific value of a reference solution consisting of water.

[0026] The aqueous polymer solution is preferably an aqueous polymer solution for cryopreservation of biological samples, the aqueous polymer solution containing a water-soluble polymer or its salt at a content of 0.1 w / v % or more and 20 w / v % or less.

[0027] The aqueous polymer solution for cryopreservation of a biological sample is preferably a water-soluble polymer containing a sugar residue.

[0028] The aqueous polymer solution for cryopreservation of the biological sample is preferably a biological sample that is a cell, a tissue, or a tissue-like substance such as a membrane or an aggregate.

[0029] The aqueous polymer solution for cryopreservation of the biological sample is preferably a biological sample selected from the group consisting of mesenchymal stem cells, blood cells, endothelial cells, and tissue for transplantation.

[0030] The aqueous polymer solution for cryopreservation of the biological sample is preferably a sperm, an egg, or a fertilized egg.

[0031] The aqueous polymer solution for cryopreservation of biological samples of the present invention is preferably used for vitrification preservation of biological samples.

[0032] The present invention also relates to a method for preventing or inhibiting the growth of ice crystals in a biological sample during cryopreservation of the biological sample by contacting the biological sample with an aqueous polymer solution for cryopreservation of the biological sample of the present invention.

[0033] The present invention also relates to a solidified body comprising a matrix containing an aqueous solvent and a water-soluble polymer or a salt thereof, and a biological sample, wherein the biological sample has an orthogonal projection area that is smaller than 8 / 10, and preferably reduced to 1 / 3.5 or more, of that in a culture solution (culture medium) before solidification, and when visible light is transmitted through the solidified body, the difference in lightness in the Munsell color system between a region occupied by the biological sample and a region occupied by the matrix is ​​3 or less, as viewed from the light exit side, and in a differential scanning calorimeter (DSC) curve of the matrix during a temperature rise process obtained under the measurement conditions of the following (1) to (2) using a DSC, the endothermic amount of an endothermic peak during the temperature rise process is 0 J / g, or 65% or less of the corresponding endothermic amount of a reference solution consisting of water. Here, the DSC scan was performed under the following conditions: (1) holding at 20°C for 1 minute, followed by cooling to -80°C at a rate of 5°C / min, and (2) holding at -80°C for 1 minute, followed by heating to 20°C at a rate of 10°C / min.

[0034] It is desirable that the intrinsic viscosity (η) of an aqueous polymer solution containing the aqueous solvent and the water-soluble polymer or a salt thereof before freezing and solidifying is 0.20 dL / g or more and 0.95 dL / g or less.

[0035] In the DSC curve during the heating process obtained under the above DSC measurement conditions (1) to (2), it is desirable for the solidified body to have no endothermic peak or to have an endothermic peak at a temperature exceeding -1.4°C and 1.1°C or lower.

[0036] In addition, "no endothermic peak is observed" means that no endothermic peak is observed in the range of -30°C to +5°C. If water containing a water-soluble polymer shows an endothermic peak, it is generally in this range.

[0037] The salt of the water-soluble polymer is preferably, for example, a metal salt, a halogen salt, or a sulfate salt. The metal salt is preferably an alkali metal or an 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 solidified material of the present invention preferably does not contain dimethyl sulfoxide, which is an intracellularly penetrating cryoprotectant for biological samples. Also, the solidified material of the present invention preferably does not contain cryoprotectants such as ethylene glycol, which are cytotoxic to biological samples, because they are harmful to biological samples after thawing.

[0039] The solidified material of the present invention is preferably a solidified frozen material (frozen solidified material).

[0040] The solidified material of the present invention preferably contains a water-soluble polymer or a salt thereof in an amount of 0.1 w / v % or more and 20 w / v % or less.

[0041] In the solidified body of the present invention, in a DSC curve during a temperature rise obtained under the above-mentioned DSC measurement conditions (1) to (2), the endothermic amount of an endothermic peak during the temperature rise is preferably 0 J / g or 40% or less of the corresponding endothermic amount of a reference solution consisting of water.

[0042] The biological sample contained in the solidified body is preferably a cell, and the cell is preferably a mammalian cell.

[0043] The biological sample contained in the solidified body is preferably a cell, and the cell is preferably a mammalian mesenchymal stem cell, a mammalian blood cell, or a mammalian endothelial cell.

[0044] The solidified body of the present invention can be preferably melted and used as a solution for administering a biological sample. The solidified body of the present invention does not contain cell-permeable and toxic compounds such as DMSO and ethylene glycol, and does not contain serum or serum-derived proteins, so that it can be used as a solution for administering a biological sample directly after melting. It is possible to add proteins that are not contaminated by bacteria or viruses. It is also possible to use a cell-permeable and toxic compound at a low concentration that does not impair the function of the cells.

[0045] It is preferable that the amount of HGF produced by the biological sample contained in a solution for administering a biological sample obtained by melting the solidified body of the present invention is suppressed compared to the amount of HGF produced by a biological sample in a solidified body containing DMSO instead of the water-soluble polymer or its salt after melting.

[0046] It is preferable that the amount of IL-10 produced by the biological sample contained in a solution for administering a biological sample obtained by melting the solidified body of the present invention is increased compared to the amount of IL-10 produced by a biological sample in a solidified body containing DMSO instead of the water-soluble polymer or its salt after melting.

[0047] In addition, it is preferable that the aqueous polymer solution and the solidified product for cryopreservation of the biological sample of the present invention do not contain cell-permeable and cytotoxic compounds such as dimethyl sulfoxide and ethylene glycol, because these are toxic to cells after thawing.

[0048] The method for cryopreserving a biological sample using the aqueous polymer solution of the present invention preferably comprises a step of preserving the biological sample by maintaining the aqueous polymer solution containing the biological sample at a temperature of -27°C or lower.

[0049] The range of the freezing and preservation temperature in the method for freezing and preserving a biological sample using the aqueous polymer solution 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.

[0050] The method for cryopreserving a biological sample is preferably one in which the step of cooling and solidifying the aqueous polymer solution containing the biological sample is carried out by a slow freezing method at a cooling rate of 10° C. / min or less. Desirably, the step of cooling and solidifying the aqueous polymer solution containing the biological sample is carried out by a slow freezing method at a cooling rate of 1° C. / min or less.

[0051] 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.

[0052] 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.

[0053] According to the aqueous polymer solution and solidified product of the present invention, biological samples such as cells and tissues can be appropriately preserved without using toxic cell-permeable compounds such as DMSO and ethylene glycol, which are intracellular-permeable cryoprotectants. Furthermore, the cryopreservation using the water-soluble polymer of the present invention is of the intracellular non-permeable type, and therefore has low toxicity to cells despite showing a high cryopreservation effect.

[0054] In addition, because it does not contain serum or serum-derived proteins, it is not contaminated by bacteria or viruses. However, it is possible to add proteins that are not contaminated by bacteria or viruses. It is also possible to use compounds that are cell-permeable and toxic at low concentrations that do not impair cell function.

[0055] The "intrinsic viscosity (η)" of the aqueous polymer solution containing a water-soluble polymer or salt used in the present invention can be determined by the following method and calculation formula.

[0056] (1) Dissolve a specified amount of NaCl in ion-exchanged water at 30°C to prepare a 0.2 M NaCl solution (standard solution). (2) Dissolve a sample of the water-soluble polymer or its salt in a standard solution at 30°C to prepare a stock solution. If the sample of the water-soluble polymer or its salt is obtained in the form of a solution, the solid portion obtained by removing the solvent from the solution is used as the sample of the water-soluble polymer or its salt. In the case of a mixed sample containing multiple water-soluble polymers, use the mixture as is as a sample. Even if the mixture contains impurities mixed with the water-soluble polymer, use it as is as a sample. At this time, 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: TIFF2025032246000001.tif2146Here, η sp : Reduced viscosity of water soluble or its salt [mL / g], η r : relative viscosity of the water-soluble polymer or its salt [-], C: concentration of the water-soluble polymer or its salt [g / mL]. (6) Plot the relationship between the concentration of the water-soluble polymer or its salt and the reduced viscosity of the water-soluble polymer or its salt, and draw an approximate line. The intercept of the approximate line (where the polymer or saccharide concentration is 0) is taken as the limiting viscosity of the water-soluble polymer or its salt.

[0057] The aqueous solvent used to dissolve the water-soluble polymer or its salt in the present invention is preferably water, a physiological solution, or an isotonic aqueous solution in which the salt concentration, sugar concentration, etc. are adjusted with sodium ions, potassium ions, calcium ions, etc. so as to be approximately the same as the osmotic pressure of a body fluid or cell fluid. Specific examples of the aqueous solvent include water, physiological saline, physiological saline having a buffering effect such as phosphate buffered saline (PBS), Dulbecco's phosphate buffered saline, Tris buffered saline (TBS), HEPES buffered saline, and balanced salt solutions such as Hank's balanced salt solution, Ringer's solution, lactate Ringer's solution, acetate Ringer's solution, and bicarbonate Ringer's solution, as well as basal culture 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, general culture media for various cells or tissues, and commercially available culture media. Effect of the Invention

[0058] According to the polymer aqueous solution and solidified body (frozen solidified body, hereinafter the solidified body will be referred to as "frozen solidified body") of the present invention, biological samples such as cells can be cryopreserved while maintaining the viability and properties of the biological samples without using highly toxic cryoprotectants such as DMSO and ethylene glycol. The water-soluble polymer or its salt, which is a cryoprotectant for the polymer aqueous solution of the present invention and is contained as a solute in the polymer aqueous solution of the present invention, is not cell membrane permeable, and therefore, during vitrification of biological samples such as cells, it does not penetrate into the biological sample and causes the biological tissue to swell, but rather promotes water removal from the cells. In the polymer aqueous solution of the present invention, the molecular species and degree of polymerization (molecular weight) of the polymer are adjusted, and / or a low molecular weight or monomolecular compound is added to the aqueous solution of the water-soluble polymer, thereby adjusting the intrinsic viscosity (η) of the polymer aqueous solution and the temperature and heat quantity of the endothermic peak of melting during the heating process. In the present invention, the effect of cryopreserving biological tissues such as cells is improved by adjusting the intrinsic viscosity (η) of the polymer aqueous solution to the range of 0.20 dL / g or more and 0.95 dL / g or less and adjusting the heat release rate of the exothermic peak measured by DSC to 0 J / g or 65% or less of the heat release rate of standard water (ultrapure water), or by adjusting the endothermic peak to 0 J / g or 65% or less of the endothermic rate of standard water (ultrapure water).

[0059] The aqueous polymer solution for cryopreservation of biological samples of the present invention is a cryopreservation solution that does not penetrate into cells, and the aqueous polymer solution and its frozen solidified form do not contain chemical substances that penetrate into cells, such as DMSO and ethylene glycol. Therefore, damage to the biological sample, cytotoxicity, and effects on cell properties as reported for DMSO can be kept low. In other words, the properties of the biological sample can be maintained during and after cryopreservation.

[0060] Furthermore, the aqueous polymer solution for cryopreservation of biological samples and the frozen solidified form thereof of the present invention make it possible to cryopreserve biological samples while maintaining their viability and properties without using serum and / or serum-derived proteins, and therefore the biological samples are not contaminated with bacteria or viruses.

[0061] Furthermore, according to the method for freezing and storing biological samples using the aqueous polymer solution of the present invention and the frozen solidified product, the frozen biological sample can be stably stored for a long period of time at a temperature of -27°C or lower, i.e., in a deep freezer, without using cytotoxic chemicals such as DMSO or ethylene glycol, or proteins such as serum. It is possible to add proteins that are not contaminated by bacteria or viruses. It is also possible to use compounds that are cell-permeable and toxic at low concentrations that do not impair the functions of cells.

[0062] 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]

[0063] [Figure 1] FIG. 1 is a graph showing the cell viability of primary human mesenchymal stem cells cryopreserved using the test sample solution of the present invention. [Figure 2A] FIG. 1 is a diagram showing the analysis results of a test sample solution by differential scanning calorimetry. [Figure 2B] FIG. 2 is an enlarged view showing the analysis results of a test sample solution by differential scanning calorimetry. [Figure 3A] FIG. 1 is a diagram showing the analysis results of a test sample solution containing ultrapure water by differential scanning calorimetry. [Figure 3B] FIG. 1 shows the analysis results of a test sample solution containing DMSO as a cryoprotectant by differential scanning calorimetry. [Figure 3C] FIG. 2 is a diagram showing the analysis results of a test sample solution containing the test sample of Example 1 by differential scanning calorimetry. [Figure 4A] FIG. 1 is a graph showing the long-term preservation effect of primary human mesenchymal stem cells cryopreserved using the test sample solution of the present invention. [Figure 4B]FIG. 1 is a graph showing the long-term preservation effect of primary human mesenchymal stem cells during refrigerated storage using the test sample 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 sample solution of the present invention. [Figure 5B] FIG. 1 is a graph showing the amount of IL-10 produced in primary human mesenchymal stem cells after cryopreservation using the test sample 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 sample solution of the present invention. [Figure 7A] FIG. 1 shows the intracellular vitrification state after freezing in a control test (Comparative Example 2). [Figure 7B] FIG. 1 shows the intracellular vitrification state after freezing using the test sample solution of Comparative Example 1. [Figure 7C] FIG. 2 shows the state of intracellular vitrification using the test sample solution of Example 1. [Figure 8A] FIG. 13 is a diagram showing the brightness difference in the intracellular vitrification state. [Figure 8B] 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 9] FIG. 1 is a graph showing the cryoprotective effect on various cells during cryopreservation using the test sample solution of the present invention. [Figure 10] FIG. 1 shows the cell viability of primary canine mesenchymal stem cells cryopreserved using low molecular weight saccharides. [Figure 11A] FIG. 1 shows the results of HPLC analysis of test samples from various production examples. [Figure 11B] FIG. 1 shows the results of HPLC analysis of test samples from various production examples. [Figure 11C] FIG. 1 shows the results of HPLC analysis of test samples from various production examples. [Figure 11D] FIG. 1 shows HPLC analysis results for a calibration curve of saccharides having a given number of sugars. [Figure 12]FIG. 2 shows the results of HPLC analysis of a test sample having a polymer chain of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0064] The aqueous polymer solution for cryopreservation of biological samples of the present invention (hereinafter also referred to as the aqueous polymer solution of the present invention) contains a water-soluble polymer or its salt, and has an intrinsic viscosity (η) in the range of 0.20 dL / g or more and 0.95 dL / g or less.

[0065] The "intrinsic viscosity (η)" of the aqueous solution containing the water-soluble polymer or its salt used in the present invention means a value calculated by the following method and formula.

[0066] 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) A sample of the water-soluble polymer or its salt is dissolved in a standard solution at 30°C to prepare a stock solution. When a sample of the water-soluble polymer or its salt is obtained in the form of a solution, the solid portion obtained by removing the solvent from the solution is used as the sample of the water-soluble polymer or its salt. In the case of a mixed sample containing multiple water-soluble polymers, the mixture is used as a sample as is. In addition, even if the water-soluble polymer contains impurities, it is used as a sample as is. However, this does not apply when measuring the viscosity average molecular weight described below. In the case of a mixed sample containing multiple water-soluble polymers, each water-soluble polymer is separated and fractionated, and the solvent is removed from each solution to use as a sample of each water-soluble polymer. In addition, if the water-soluble polymer is unknown, the substance is identified by HPLC, LC-MS, LC-IR, etc. for the water-soluble polymer, and the viscosity average molecular weight is calculated as described below. In addition, if the water-soluble polymer contains multiple unknown water-soluble polymers, each component is separated and fractionated, and the substance is identified by HPLC, LC-MS, LC-IR, etc. for each water-soluble polymer, and the viscosity average molecular weight is calculated as described below. In addition, even if the mixture contains impurities mixed in with the water-soluble polymer, if the viscosity is not affected (for example, impurities such as metal salts), the mixture is used as the sample of the water-soluble polymer. If the solution contains impurities that affect the calculation of the viscosity average molecular weight, remove the impurities or fractionate the water-soluble polymer before measurement. Measure the viscosity of the standard solution and the stock solution, and adjust the relative viscosity of the stock solution to 2.0 to 2.4 compared to the standard solution. (3) Dilute the 30°C stock solution 5 / 4, 5 / 3, and 5 / 2 times with the 30°C standard solution. (4) Measure the viscosity of the standard solution, undiluted solution, and diluted solution at 30°C. Use an E-type viscometer to measure the viscosity. (5) The viscosity of the original solution and the diluted solution divided by the viscosity of the standard solution is the relative viscosity (η r ) and derive the reduced viscosity based on the following formula: TIFF2025032246000002.tif2146Here, η sp : Reduced viscosity of water soluble or its salt [mL / g], η r : relative viscosity of the water-soluble polymer or its salt [-], C: concentration of the water-soluble polymer or its salt [g / mL]. (6) Plot the relationship between the concentration of the water-soluble polymer or its salt and the reduced viscosity of the water-soluble polymer or its salt, and draw an approximate line. The intercept of the approximate line (where the polymer or saccharide concentration is 0) is taken as the limiting viscosity of the water-soluble polymer or its salt.

[0067] The polymer aqueous solution of the present invention is a cryopreservation solution for appropriately preserving a biological sample when the biological sample is cryopreserved. By setting the intrinsic viscosity (η) of the polymer aqueous solution of the present invention to be in the range of 0.20 dL / g or more and 0.95 dL / g or less, the polymer aqueous solution of the present invention can trap aqueous solvent molecules well in the matrix formed by the polymer chains during the cooling process. This restricts the molecular motion of water in the aqueous solvent during cooling, and allows water to be solidified and / or frozen in a vitrified state without crystallizing. A freezing method called a vitrification method is a method in which, when a solution is frozen, a solute (a cryoprotectant for preventing freezing damage) is excluded from the crystals, increasing the salt concentration in the remaining solution, and an osmotic pressure difference occurs between the inside and outside of the cell, dehydrating the inside of the cell and vitrifying the inside of the cell, and is applied to cells with a particularly low survival rate after thawing. In such a method, in order to make water easier to vitrify, the concentration of the solute (cryoprotectant) is increased or the cooling rate is increased. However, it has been known that increasing the osmotic pressure difference increases damage to cells, and that recrystallization occurs during thawing, causing damage to cells. There are also technical difficulties involved.

[0068] When the polymer aqueous solution of the present invention is used as a cryopreservation solution for biological samples such as cells, the action of the polymer chains of the water-soluble polymer dissolved in the polymer aqueous solution causes the inside of the biological sample to be dehydrated and vitrified, suppressing or preventing the formation of ice crystals in the biological sample and further reducing the osmotic shock in the biological sample during freezing, which is a problem in conventional vitrification methods.Therefore, there is no need to contain chemical substances such as dimethyl sulfoxide (DMSO) or ethylene glycol that have cytotoxicity, and the polymer aqueous solution of the present invention does not contain DMSO and / or ethylene glycol.

[0069] In the aqueous polymer solution containing the water-soluble polymer of the present invention, the water molecules of the solvent are considered to exist in the network matrix of the polymer during freezing, and to exist in the state of free water or bound water (a state in which the water molecules are entangled and captured by the polymer). In addition, it is considered that the water molecules that have been expelled from inside the cells, etc. due to the osmotic pressure difference are replaced with the polymer around the biological sample such as the cell. In order to realize this state, it is necessary to adjust the intrinsic viscosity of the aqueous polymer solution to 0.20 to 0.95 dL / g. If the intrinsic viscosity of the aqueous polymer solution is too small, the polymer will diffuse, and the water molecules will not be entangled and captured in the network of the water-soluble polymer. On the other hand, if the intrinsic viscosity of the aqueous polymer solution is too high, the replacement with the water molecules near the biological sample will not proceed. In addition, a problem may occur in that the handling property is deteriorated when the biological sample is contained in the aqueous polymer solution for cryopreservation of the biological sample. By setting the intrinsic viscosity of the aqueous polymer solution to the range of 0.20 dL / g or more and 0.95 dL / g or less, when the aqueous polymer solution of the present invention is cooled, the amorphous glass state in the frozen state is stabilized. Therefore, cells are less likely to be damaged by cooling and freezing, and can be cryopreserved stably and efficiently. Cell destruction due to ice crystal formation is also less likely to occur. Therefore, after thawing the cryopreserved biological sample of the present invention, the survival rate of cells in the biological sample is high. For example, it may be preferable that the intrinsic viscosity (η) of the aqueous polymer solution is about 0.32 dL / g or more and 0.65 dL / g or less.

[0070] The intrinsic viscosity of a polymer aqueous solution can be adjusted by selecting the molecular species and degree of polymerization (viscosity average molecular weight) of the water-soluble polymer.

[0071] For example, the water-soluble polymer in the present invention may be a polymer containing a monomer having a hydrophilic group as a repeating unit. Here, the hydrophilic group is, for example, a hydroxyl group and a carboxylic acid group and a salt thereof. The water-soluble polymer may also contain a nitrogen-containing monomer having an amino group which may be substituted or an amide group which may be substituted as a repeating unit. In particular, it may be preferable for the water-soluble polymer to have a hydroxyl group at an equatorial position in its structure. This is because it is believed that the water of the solvent can be more effectively trapped in the matrix formed by the polymer chains during freezing.

[0072] The monomer having a hydrophilic group is, for example, a sugar residue. In this case, the polymer in the present invention may be a water-soluble polymer including a polymer in which sugar residues are bonded as repeating units through glycosidic bonds and derivatives thereof. Examples of sugar residues include, but are not limited to, monosaccharides, or monosaccharides in which the hydroxyl and / or hydroxymethyl groups of the monosaccharides are substituted, for example, monosaccharides in which the hydroxyl and / or hydroxymethyl groups are substituted with at least one substituent selected from the group consisting of a carboxyl group, an amino group, an N-acetylamino group, a sulfoxy group, a methoxycarbonyl group, and a carboxymethyl group.

[0073] Examples of monosaccharides include triose, tetrose, pentose, hexose, and heptose. For example, examples of pentoses include ribose, arabinose, xylose, lyxose, xylulose, ribulose, and deoxyribose. Examples of hexoses include glucose, mannose, galactose, fructose, sorbose, tagatose, fucose, fuculose, and rhamnose.

[0074] For example, the monosaccharide substituted with a carboxyl group includes uronic acid. Examples of the uronic acid include glucuronic acid, iduronic acid, mannuronic acid, and galacturonic acid. Examples of the monosaccharide substituted with an amino group include amino sugars. Examples of the amino sugar include glucosamine, galactosamine, mannosamine, and muramic acid. Examples of the monosaccharide substituted with an N-acetylamino group include N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, and N-acetylmuramic acid. Examples of the monosaccharide substituted with a sulfooxy group include galactose-3-sulfate. Examples of the monosaccharide having multiple substituents include N-acetylglucosamine-4-sulfate, iduronic acid-2-sulfate, glucuronic acid-2-sulfate, N-acetylgalactosamine-4-sulfate, neuraminic acid, and N-acetylneuraminic acid.

[0075] For example, the water-soluble polymer of the present invention is a polymer containing the above-mentioned monosaccharide as a repeating unit. For example, the water-soluble polymer of the present invention may be a polymer containing pentose, hexose, or uronic acid, which may be substituted, or a combination thereof as a repeating unit. For example, the water-soluble polymer of the present invention may contain a six-membered ring structure in its main chain. This is because polysaccharides having a six-membered ring structure in their main chain may easily form a polymer network structure around cells through hydrogen bonds. In addition, the water-soluble polymer of the present invention may be an alternating copolymer of a monomer having a hydrophilic group and a nitrogen-containing monomer. The nitrogen-containing monomer may be, for example, an amino sugar. In this case, for example, the water-soluble polymer of the present invention may be a glycosaminoglycan. It may also be a sulfated polysaccharide in which one or more hydroxyl groups are substituted with sulfooxy groups. Although not limited thereto, examples of polymers that can be used in the present invention include hyaluronic acid, dextran, pullulan, and chondroitin sulfate.

[0076] The water-soluble polymer or its salt used in the present invention may be naturally derived or may be chemically synthesized. A commercially available water-soluble polymer or its salt may be used as it is. A naturally derived polymer compound or a commercially available polymer compound having a larger molecular weight may be used, and the cleavage product may be obtained by subjecting it to hydrolysis, enzyme treatment, subcritical treatment, or the like, and the molecular weight may be adjusted to form the water-soluble polymer of the present invention. In addition, each monomer may be naturally derived, may be modified or substituted to be used, or may be chemically synthesized. For example, preferably, the monomer contained in the water-soluble polymer of the present invention is a biological component. It is considered that the cytotoxicity is low when a polymer aqueous solution containing a water-soluble polymer is used as a cryopreservation solution for a biological sample. In addition, a process required for a conventional cryopreservation solution, such as washing and removing a cryoprotectant contained in the cryopreservation solution from a thawed biological sample, may be unnecessary or simplified.

[0077] The water-soluble polymer or its salt in the present invention has a viscosity-average molecular weight of, for example, more than 3000 and 500000 or less. By using a water-soluble polymer or its salt having such a viscosity-average molecular weight, when the aqueous solution is cooled, the amorphous glass state in the frozen state of the solvent is stabilized. Therefore, cells are less likely to be damaged by cooling and freezing, and cells can be 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 water-soluble polymer is 3000 or less, vitrification may not occur well. In addition, if the viscosity-average molecular weight of the water-soluble polymer is more than 500000, the viscosity increases significantly, and the solubility decreases, or the prepared solution foams, which may cause problems such as poor handling. The viscosity-average molecular weight is preferably, for example, 10000 or more. In addition, polymers having a viscosity-average molecular weight of 400000 or less, or even 200000 or less, are preferable, and 150000 or less is particularly preferable. This is because the viscosity of the aqueous solution can be adjusted to be low, and the solution is easy to handle.

[0078] The "viscosity average molecular weight" of the water-soluble polymer or salt used in the present invention is determined by the following method and formula using the intrinsic viscosity (η) calculated by the above method and formula.

[0079] Viscosity average molecular weight: The viscosity average molecular weight is calculated from the intrinsic viscosity. TIFF2025032246000003.tif3559The viscosity average molecular weight M is calculated from the intrinsic viscosity determined by measurement and the K and α values ​​published in the literature, etc., in the Mark Hoying-Sakurada formula above. For example, if the water-soluble polymer is hyaluronic acid, K = 3.6 × 10 -4 and α = 0.78. In the case of pullulan and gelatin, K = 9 × 10 -4 , α=0.5, and for dextran, K=6.3×10 -8 , α = 1.4, and for chondroitin sulfate, K = 5.8 × 10 -4 , α = 0.74, for fructan (levan type), K = 1.6 × 10 -3 , α = 0.45, and carboxylated polylysine has a K = 2.78 × 10 -5 , α=0.87.

[0080] K and α are values ​​that vary depending on the type of polymer. For example, the values ​​of K and α are disclosed in many published documents, such as the "Polymer Materials Handbook" (edited by the Society of Polymer Science). Using these published values, the viscosity average molecular weight can be calculated from the intrinsic viscosity (η).

[0081] In the present invention, the molecular weight calculated by this method is regarded as the viscosity average molecular weight. In the case of monosaccharides and disaccharides such as sucrose and glucuronic acid, and compounds considered to be monomolecular, the molecular weight is clearly specified from the structural formula, so the molecular weight specified from the structural formula is treated as the viscosity average molecular weight.

[0082] The hydrophilic groups of the water-soluble polymer or its salt used in the present invention are preferably unmodified or, even if modified, are less than 50% of the total number of hydrophilic groups, i.e., no substituents are introduced into the polymer chain or, even if they are introduced, are less than 50% of the total number of hydrophilic groups. It is presumed that the hydrophilic groups of the water-soluble polymer, especially the OH group, NH2 group, and COOH group, contribute to the protection of the biological sample, the vitrification of the solvent, and the replacement with water molecules around the biological sample, so that it is advantageous to improve the survival rate of the biological sample if these functional groups are not modified. Furthermore, when a functional group is introduced into the water-soluble polymer, the reagent used to modify the hydrophilic group of the polymer may adversely affect the cryoprotective effect of the polymer or may make it impossible to use it as a cryopreservation solution for cryopreservation of biological samples for regenerative medicine purposes.

[0083] The salt of the water-soluble polymer used in the present invention may be a metal salt, a halogen salt, or a sulfate salt. The metal salt is preferably an alkali metal or an 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.

[0084] As the aqueous solvent for the aqueous polymer solution of the present invention, water, a physiological solution, or an isotonic aqueous solution in which the salt concentration or sugar concentration is adjusted by sodium ions, potassium ions, calcium ions, etc. so as to be approximately the same as the osmotic pressure of a body fluid or a cell fluid is preferred. Specifically, for example, water, physiological saline, physiological saline having 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 an isotonic agent, a chelating agent, and a solubilizing agent, so long as the effects of the present invention are not impaired. In this specification, the term "optional component" means a component that may or may not be contained. For example, the solvent may be a 5% glucose aqueous solution. In addition, a medium for cell culture may be used as the aqueous solvent for preparing the aqueous polymer 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, M-199, and general culture fluids for various cells or tissues. Therefore, when a biological sample is cryopreserved using the aqueous polymer solution of the present invention, a biological sample such as cells after culture may be suspended in the aqueous polymer solution of the present invention and cryopreserved, or the water-soluble polymer or a salt thereof of the present invention may be added at a desired concentration to a culture fluid containing a biological sample such as a cell culture fluid or a cell suspension after culture, and the cryopreservation step may be performed.

[0085] In addition, the aqueous polymer solution of the present invention may be pH adjusted as necessary. For example, when the hydrophilic group of the monomer having a hydrophilic group is a carboxylic acid group, the aqueous polymer solution containing the water-soluble polymer polymerized from such a monomer may be acidic. In such a case, the aqueous polymer solution may be made neutral by adjusting the pH, which may make the solution more suitable for the survival of the cells to be cryopreserved, and the survival rate of the cells may be improved. There is no limitation on the salt used for adjusting the pH, and any salt commonly used for adjusting the pH of an aqueous solution may be used.

[0086] According to the results of thermal analysis of the polymer aqueous solution of the present invention by differential scanning calorimetry (DSC), the polymer aqueous solution of the present invention exhibits the following characteristics (1) and (2): In a DSC curve during a temperature drop obtained under fixed conditions, the heat release value of the exothermic peak during the temperature drop is 0 J / g or is 65% or less of the corresponding heat release value of a reference solution made of water, preferably 40% or less of the corresponding heat release value of a reference solution made of water. DSC conditions: (1) Hold at 20°C for 1 minute, then cool to -80°C at a rate of 5°C / min, and (2) After holding at -80°C for 1 minute, increase the temperature to 20°C at a rate of 10°C / min.

[0087] The fact that the heat value of the exothermic peak during the temperature drop process in the polymer aqueous solution of the present invention is smaller (less than 100%) than the corresponding heat value of the reference solution made of water in the DSC measurement means that bound water and free water that do not form ice crystals remain in the frozen polymer aqueous solution. That is, in the polymer aqueous solution of the present invention, the replacement of water with the water-soluble polymer and / or low molecular weight saccharide proceeds, and water molecules are trapped in the matrix of the water-soluble polymer, restricting the molecular motion of water. This promotes dehydration of water from within the cells, and allows water to be solidified in a glassy state without crystallization. Good vitrification is achieved in the polymer aqueous solution and the biological sample contained therein. Therefore, in the cryopreservation of a biological sample using the polymer aqueous solution of the present invention, destruction of cells due to the formation of ice crystals can be suppressed or prevented. Furthermore, in the polymer aqueous solution of the present invention, not only is the formation of ice crystals suppressed or prevented during the cooling process, but the recrystallization of water during the subsequent temperature rise process, i.e., during melting, can also be suppressed. That is, in the polymer aqueous solution of the present invention, the glassy state of the biological sample in the frozen state is considered to be stabilized. Since the glassy state is further stabilized in this way, the aqueous polymer solution of the present invention exhibits a high cell protection effect without the need for the addition of protein components such as serum derived from humans or cows or serum-derived components (e.g., albumin, etc.). Therefore, there is no need to worry about infectious diseases, and it is considered that the solution is not affected by lot-to-lot differences in biological preparations. It is possible to add proteins that are not contaminated by bacteria or viruses.

[0088] In addition, in the solidified body (frozen solidified body) of the present invention, the result of thermal analysis of the matrix of the frozen solidified body of the present invention by differential scanning calorimetry (DSC) shows that in the matrix contained in the frozen solidified body of the present invention, in a DSC curve for the temperature rise process obtained under the measurement conditions of the following (1) to (2), the endothermic heat of the endothermic peak during the temperature rise process is 0 J / g or is 65% or less of the endothermic heat corresponding to the reference solution consisting of water, desirably 40% or less of the endothermic heat corresponding to the reference solution consisting of water. DSC conditions: (1) Hold at 20°C for 1 minute, then cool to -80°C at a rate of 5°C / min, and (2) After holding at -80°C for 1 minute, increase the temperature to 20°C at a rate of 10°C / min.

[0089] The fact that the endothermic heat of the endothermic peak during the temperature rise process in the matrix of the present invention is smaller (less than 100%) than the corresponding endothermic heat of the reference solution made of water means that bound water and free water that do not form ice crystals remain in the frozen matrix. That is, in the aqueous solution of the water-soluble polymer forming the matrix of the present invention, the replacement of the water-soluble polymer and / or low molecular weight saccharide with water proceeds, and water molecules are trapped in the matrix of the water-soluble polymer, restricting the molecular motion of water. This promotes dehydration of water from within the cells of the biological sample contained in the matrix, and allows water to be solidified in a glassy state without crystallization. Good vitrification of the matrix in the frozen solidified body and the inside of the biological sample contained in the frozen solidified body is achieved. Therefore, in the frozen solidified body of the present invention, destruction of cells due to the formation of ice crystals can be suppressed or prevented. Furthermore, in the frozen solidified body of the present invention, not only is ice crystal formation suppressed or prevented during the cooling process, but recrystallization of water during the subsequent temperature rise process, i.e., during melting, can also be suppressed. That is, in the frozen solidified body of the present invention, it is considered that the glassy state of the biological sample contained in the frozen solidified body is stabilized in the frozen state. Since the glassy state is further stabilized in this way, the biological sample stored in the frozen solidified body of the present invention exhibits a high cell protection effect without the need for the addition of protein components such as serum derived from humans or cows or serum-derived components (e.g., albumin, etc.). Therefore, it is considered that the biological sample after thawing is free from concerns about infectious diseases and is not affected by lot-to-lot differences in biological preparations. It is possible to add proteins that are not contaminated by bacteria or viruses.

[0090] The amount of heat generated and the amount of heat absorbed can be adjusted by selecting the molecular species of the water-soluble polymer and / or adjusting the amount added. For example, hyaluronic acid has a small amount of heat generated and an endothermic heat, while pullulan has a large amount of heat generated and an endothermic heat. In addition, increasing the content of the water-soluble polymer reduces both the amount of heat generated and the amount of heat absorbed.

[0091] In the aqueous polymer solution for cryopreservation of biological samples of the present invention, in a DSC curve during a temperature rise obtained using a differential scanning calorimeter (DSC) under the measurement conditions of the following (1) to (2), it is desirable that no endothermic peak is observed or that the temperature at which an endothermic peak is observed is higher than -1.4°C and 1.1°C or lower. DSC conditions: (1) Hold at 20°C for 1 minute, then cool to -80°C at a rate of 5°C / min, and (2) After holding at -80°C for 1 minute, increase the temperature to 20°C at a rate of 10°C / min.

[0092] That is, in the polymer aqueous solution of the present invention, the endothermic peak presumably due to the melting of water is not observed during the temperature rise process of the polymer aqueous solution, or if observed, it is in the range of more than -1.4°C and less than 1.1°C, that is, near the temperature (0.7°C) at which an endothermic peak is observed in ultrapure water in DSC measurement under the same conditions. For vitrification in a biological sample such as a solvent or a cell, it generally seems desirable to use a cryopreservation solution with a lower melting point (freezing point). However, the present inventors have found that for good vitrification of a biological sample during freezing, it is desirable for the cryopreservation solution to have the opposite characteristic to this technical common sense. Usually, the phenomenon of a melting point (freezing point) being lower than that of water occurs when a solute is dispersed in a water matrix, but in the polymer aqueous solution of the present invention, on the contrary, water molecules are present in the polymer network matrix in the aqueous solvent formed by the water-soluble polymer, and the water does not freeze in the matrix but exists as free water, or even if the water freezes, it freezes in a state where it is entangled in the polymer (bound water). For this reason, in the aqueous polymer solution of the present invention, it is presumed that no endothermic peak due to the melting of water is observed during the temperature increase process, or that the endothermic peak is observed at a temperature close to that at which the endothermic peak of pure water is observed.

[0093] Furthermore, the present inventors have found that in the present invention, the endothermic peak can be adjusted by adding a saccharide or a salt thereof having a lower molecular weight than the water-soluble polymer to an aqueous polymer solution and a frozen solid containing the water-soluble polymer or a salt thereof in the present invention.

[0094] The low molecular weight saccharide or its salt is held in the polymer matrix by hydrogen bonding due to the hydrophilic group in the water-soluble polymer of the present invention. It is believed that the presence of a polymer holding a low molecular weight saccharide or its salt around a biological sample such as a cell replaces water molecules near the cell membrane with the low molecular weight saccharide. Therefore, it is believed that the water discharged from the biological sample such as a cell to the outside of the biological sample due to the osmotic pressure difference during freezing will replace not only the polymer around the biological sample such as a cell, but also the low molecular weight saccharide. Therefore, it is believed that the addition of the low molecular weight saccharide or its salt helps to discharge water from the biological sample, and by replacing it with water molecules, the formation and growth of ice crystals near the cell membrane is suppressed or prevented. By setting the intrinsic viscosity of the aqueous polymer solution of the present invention to a range of 0.20 dL / g or more and 0.95 dL / g or less, it is possible to promote the replacement of water molecules with low molecular weight saccharides. As a result, cell membrane damage during freezing can be significantly suppressed. In other words, the low molecular weight saccharide or its salt can function as a component for protecting cells in the aqueous polymer solution and the frozen solidified body of the present invention. If the intrinsic viscosity of the aqueous polymer solution of the present invention is too small, the low molecular weight saccharides will diffuse in the aqueous polymer solution, and the water molecules will not be efficiently entangled and captured in the water-soluble polymer network.On the other hand, if the intrinsic viscosity of the aqueous polymer solution is too high, it is considered that the exchange of the low molecular weight saccharides with water molecules will not proceed efficiently.

[0095] Since the discharge of water from the biological sample is promoted and the cells are protected in this way, the volume of the biological sample, such as cells, frozen in the aqueous polymer solution of the present invention (i.e., frozen in a frozen solid) shrinks compared to the biological sample before freezing. For example, the orthogonal projection area of ​​the biological sample in the aqueous polymer solution of the present invention solidified at -80°C is reduced to about 1 / 3.5 or more and less than 8 / 10 compared to the orthogonal projection area of ​​the biological sample in the culture medium (in the culture medium) before cooling (before freezing). When the biological sample is frozen in the aqueous polymer solution of the present invention, the inside of the cells is well dehydrated during freezing. This significantly reduces damage to the cells during freezing, and the thawed cells show a high survival rate.

[0096] In the present invention, the "orthogonal projection area" of a biological sample refers to the area calculated by taking an image of the biological sample from above in the normal direction of the cooling stage when the biological sample is observed on a cooling stage for a microscope, and calculating the area of ​​the biological sample in the image using the image analysis software ImageJ (https: / / imagej.nih.gov / ij / ).

[0097] In order to retain low molecular weight saccharides or their salts in the matrix, it is desirable that the hydrophilic groups of the water-soluble polymer or its salt used in the present invention are not modified, or even if modified, the total number of hydrophilic groups is 50% or less, that is, no substituent is introduced into the polymer chain, or even if it is introduced, the total number of hydrophilic groups is 50% or less. If the hydrophilic group of the water-soluble polymer is modified, the retention effect of the low molecular weight saccharides or their salts by the hydrophilic group is reduced, and even if low molecular weight saccharides coexist, it may not be possible to fully contribute to improving the survival rate of the biological sample. Therefore, for example, it may not be preferable to modify the OH group or NH2 group of the water-soluble polymer with a carboxylic acid or the like.

[0098] The low molecular weight saccharide or its salt in the present invention is, for example, a saccharide or its salt having a viscosity average molecular weight of 3000 or less. Preferably, it may be a monosaccharide, disaccharide, oligosaccharide, or the like having a viscosity average molecular weight of 1000 or less.

[0099] The low molecular weight saccharide in the present invention may be, for example, the monosaccharide described above as the monomer constituting the water-soluble polymer in the present invention. For example, the low molecular weight saccharide is glucose, fructose, galactose, or uronic acid in which the alcohol group of these is oxidized, or an amino sugar in which the alcohol group is substituted with an amino group, sucrose, trehalose, or a polymer or combination thereof. In addition, the low molecular weight saccharide may be, for example, a fragment of a polymer, such as hyaluronic acid, dextran, pullulan, or chondroitin sulfate. Although not particularly limited as long as it does not impair the effect of the present invention, the low molecular weight saccharide may be, for example, a cleavage product (fragment) of glycosaminoglycan, that is, a monosaccharide constituting glycosaminoglycan, a disaccharide thereof, or an oligosaccharide thereof. In the case of hyaluronic acid, it is preferable to use one having a viscosity average molecular weight of 1000 or less.

[0100] Preferably, low molecular weight saccharide is the cleavage product of hyaluronic acid.Therefore, preferably, low molecular weight saccharide or its salt in the present invention is glucuronic acid or N-acetylglucosamine, or the disaccharide or oligosaccharide thereof, or its salt.Preferably, saccharide can be glucuronic acid or its modified compound, or its disaccharide or oligosaccharide, or its salt.

[0101] In the present invention, the "cleavage product" refers to a compound having a smaller molecular weight than the original polymer, which is considered to be obtained when a polymer is subjected to hydrolysis, enzyme treatment, subcritical treatment, or the like. For example, the polymer used in the present invention may be a water-soluble polymer obtained by treating a larger polymer compound as described above, and the low molecular weight saccharide used in the present invention may be a low molecular weight saccharide obtained by treating a water-soluble polymer. In the present invention, the cleavage product may be a monomer that is a component of the original polymer and / or a polymer of various polymerization degrees of the monomer and / or a mixture thereof.

[0102] "Subcritical processing" means contacting the raw material to be extracted with a subcritical fluid as an extracting solvent that has been brought into a subcritical state under conditions of a predetermined temperature and a predetermined pressure. For example, when water is raised to a pressure of 22.12 MPa or more and a temperature of 374.15°C or more, it is in a state that is neither liquid nor gas. This point is called the critical point of water, and hot water with a temperature and pressure close to the critical point is called subcritical water. Using the hydrolysis action of this subcritical water, the desired components can be obtained from the raw material to be extracted. In the present invention, the conditions for subcritical processing are, for example, a temperature of 150°C or more and 350°C or less, and the subcritical processing pressure can be equal to or higher than the saturated vapor pressure at each temperature, but can be, for example, 0.5 MPa or more and 25 MPa or less. After the subcritical processing, components with a molecular weight of less than a predetermined value are separated and collected, and can be used as the cleavage product in the present invention. In addition, there are no particular limitations on the hydrolysis or enzyme processing, and reagents and processing methods that are commonly used can be used without problems.

[0103] The water-soluble polymer and low molecular weight saccharides of the present invention may be obtained simultaneously by a single subcritical treatment. That is, the water-soluble polymer and low molecular weight saccharides used in the present invention may be subcritically treated products of polymer compounds having a first molecular weight distribution in a molecular weight range of more than 3000 and not more than 500000 as a viscosity average molecular weight, and a second molecular weight distribution in a molecular weight range of not more than 3000 as a viscosity average molecular weight. Therefore, for example, the method for producing an aqueous polymer solution of the present invention may include a step of obtaining the water-soluble polymer and low molecular weight saccharides of the present invention by dissolving a polymer, which is a polysaccharide having a molecular weight of more than 500000 as a viscosity average molecular weight, in water and then performing an extraction treatment under subcritical conditions of water. However, the water-soluble polymer and low molecular weight saccharides of the present invention may be used in combination after being treated and / or separated based on molecular weight in separate treatment steps.

[0104] The low molecular weight saccharide or its salt in the present invention has an endothermic peak at a temperature lower than the temperature (0.7℃) at which the endothermic peak is observed in pure water by DSC measurement.For example, the hyaluronic acid with viscosity average molecular weight of 1000 or less has an endothermic peak temperature of -8.1℃, which is lower than the endothermic peak temperature of pure water, 0.7℃.

[0105] On the other hand, the water-soluble polymer or its salt in the present invention has, for example, hyaluronic acid with a viscosity average molecular weight of 15000, an endothermic peak temperature of -1.4°C, and pullulan with a viscosity average molecular weight of 373000, a temperature of 1.49°C. When low molecular weight saccharide or its salt is combined with such a water-soluble polymer or its salt, the endothermic peak temperature of the polymer aqueous solution is brought close to the endothermic peak temperature of pure water, or the endothermic peak itself is not observed in the measurement by DSC.For example, when hyaluronic acid with a viscosity average molecular weight of 1000 or less is added to hyaluronic acid with a viscosity average molecular weight of 15000, the endothermic peak temperature does not decrease, but increases to -0.4°C.On the other hand, when hyaluronic acid with a viscosity average molecular weight of 1000 or less is added to pullulan with a viscosity average molecular weight of 373000, the endothermic peak temperature decreases to 0.83°C.

[0106] That is, when the hyaluronic acid with viscosity average molecular weight of 1000 or less is mixed with the water-soluble polymer whose endothermic peak temperature is lower than that of pure water, the endothermic peak temperature rises and approaches the endothermic peak temperature of pure water, or the endothermic peak itself does not appear in DSC.In addition, when the hyaluronic acid with viscosity average molecular weight of 1000 or less is mixed with the water-soluble polymer whose endothermic peak temperature is higher than that of pure water, the endothermic peak temperature drops and approaches the endothermic peak temperature of pure water, or the endothermic peak itself does not appear in DSC.In this way, for example, by using the hyaluronic acid with viscosity average molecular weight of 1000 or less, the endothermic peak temperature of water-soluble polymer can be adjusted to be around the endothermic peak temperature of pure water, which is 0.7℃.

[0107] The low molecular weight saccharide or its salt that has such an effect is not limited to the hyaluronic acid with the viscosity average molecular weight of 1000 or less. As long as the viscosity average molecular weight is 1000 or less, other saccharides such as sucrose, trehalose, etc., other than hyaluronic acid, can have the same effect. It is speculated that the low molecular weight saccharide used in the present invention exists between water molecules and inhibits water from crystallizing, or cooperates with the water-soluble polymer network to trap water in the network (either bound water or free water), inhibiting water from crystallizing in the first place, so that the polymer aqueous solution and frozen solid of the present invention can achieve the behavior close to that of pure water.

[0108] Since the polymer aqueous solution and the frozen solid of the present invention do not contain a compound that penetrates cells, it is considered that the cytotoxicity is low when the polymer aqueous solution and the frozen solid of the present invention are brought into contact with a biological sample for cryopreservation of the biological sample. Furthermore, since it is not necessary to increase the concentration of solutes to restrict the molecular motion of water, it is considered that the damage to the cells is low. In addition, the low molecular weight sugar or its salt added has a cytoprotective effect of quickly replacing the water discharged from the inside of the cells around the cells during freezing, so it is considered that the properties of the cells are not changed during cryopreservation. Therefore, it is considered that the polymer aqueous solution and the frozen solid of the present invention can freeze and preserve the biological sample while maintaining the properties of the cells.

[0109] In addition, unlike conventional vitrification methods, the aqueous polymer solution and the frozen solidified product of the present invention do not require a high cooling rate during cooling to reduce osmotic shock. Therefore, the aqueous polymer solution and the frozen solidified product of the present invention enable efficient cryopreservation of biological samples in a simple manner with reduced toxicity, and after cryopreservation, thawed cells show a high survival rate.

[0110] The aqueous polymer solution and frozen solid of the present invention contain a water-soluble polymer or a salt thereof at a concentration of about 0.1 w / v% or more and 20 w / v% or less. If the concentration of the water-soluble polymer is too low, the solvent portion may not be vitrified well. For example, the concentration of the water-soluble polymer or a salt thereof is preferably 1 w / v% or more, and particularly preferably 5 w / v% or more. In addition, if the concentration is higher than 20 w / v%, the viscosity may become too high, which may deteriorate the handling properties. Preferably, the concentration of the polymer or a salt thereof is 5 w / v% or more and 20 w / v% or less.

[0111] The aqueous polymer solution and the frozen solid of the present invention preferably contain low molecular weight saccharides or salts thereof such that the content ratio of the water-soluble polymer to the low molecular weight saccharide in the aqueous polymer solution is about 10:1. If the concentration of the saccharides or salts thereof is too low, the cell protection effect of the low molecular weight saccharides may not be sufficiently obtained. In addition, even if the saccharides are added at a concentration of 1 / 10 or more of the water-soluble polymer, it is difficult to obtain further effects as a cell protection component.

[0112] According to the results of thermal analysis of the polymer aqueous solution and the frozen solidified product of the present invention by differential scanning calorimetry (DSC), it is desirable that the polymer aqueous solution and the frozen solidified product of the present invention have a glass transition temperature in the vicinity of -23°C ± 4°C. In the polymer aqueous solution and the frozen solidified product of the present invention, as described above, vitrification is achieved by entangling and capturing water molecules in the network of the water-soluble polymer during freezing. Therefore, a large cooling rate for cell protection during cooling, which is required in conventional vitrification methods, is not required. For this reason, after a biological sample is immersed in the polymer aqueous solution of the present invention, it is cooled to -27°C or lower, which is the glass transition temperature of the polymer aqueous solution of the present invention, to form a frozen solidified product. For example, cells, tissues, etc. to be preserved can be stably frozen and preserved by simply placing the polymer aqueous solution of the present invention containing a biological sample in a freezing treatment container or the like and leaving it in a deep freezer at -80°C. 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 a special container for liquid nitrogen cryopreservation or the preparation of liquid nitrogen. Therefore, the operation related to the cryopreservation of biological samples can be significantly simplified. In addition, the aqueous polymer solution and the frozen solid of the present invention can suppress the recrystallization of water during thawing, so that the aqueous polymer solution of the present invention can easily and efficiently perform a series of steps of freezing, preserving, and thawing a biological sample without requiring special procedures. Of course, it is also possible to freeze and preserve a biological sample in liquid nitrogen or liquid nitrogen vapor using the aqueous polymer solution of the present invention and liquid nitrogen.

[0113] Since the polymer aqueous solution and the frozen solidified product of the present invention function as a non-permeable cryopreservation liquid and a frozen solidified product obtained by freezing the cryopreservation liquid, the biological sample to be cryopreserved using the polymer aqueous solution and the frozen solidified product of the present invention is not particularly limited. They can be used for cryopreservation of various types of cells. In addition, the origin of the cells is not particularly limited. The polymer aqueous solution and the frozen solidified product of the present invention can effectively suppress or prevent ice crystal formation and recrystallization during freezing and thawing, and therefore can be used well for mammalian cells having complex structures. Therefore, they can be applied to the cryopreservation of cells of various animal species, including mice, dogs, and humans. Furthermore, the polymer aqueous solution and the frozen solidified product of the present invention are known to be more damaged during freezing than general culture cells, and can be particularly suitably used for the cryopreservation of reproductive cells such as stem cells, early embryos, eggs, sperm, and fertilized eggs, which are inevitably subject to a significant decrease in survival rate by the so-called conventional slow freezing method. Furthermore, since the polymer aqueous solution and frozen solid of the present invention do not contain chemical substances such as DMSO or ethylene glycol that can act as differentiation factors, they can be used to preserve cells that need to remain undifferentiated. For example, stem cells for regenerative medicine applications can be frozen and preserved without the risk of differentiation.

[0114] In addition, because serum or serum-derived proteins are not added, there is no contamination with bacteria or viruses. However, it is possible to add proteins that are not contaminated by bacteria or viruses. In addition, it is possible to use compounds that are cell-permeable and toxic at low concentrations that do not impair cell function.

[0115] Furthermore, since the aqueous polymer solution and frozen solid of the present invention have an excellent effect of stabilizing the glassy state in a frozen state, they 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 substances, such as tissues obtained through regenerative medicine.

[0116] That is, the polymer aqueous solution and the frozen solidified product of the present invention can be used for a biological sample selected from cells, tissues, or tissue-like substances such as membranes or aggregates, and can realize a high survival rate. In particular, the polymer aqueous solution and the frozen solidified product of the present invention can be advantageously used in the 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, in the cryopreservation of stem cells of primates, which are considered to have lower freeze resistance than mice, the cryopreservation of tissues for transplantation, and the cryopreservation of germline cells in reproductive medicine.

[0117] In addition, if the water-soluble polymer in the aqueous polymer solution and the frozen solid of the present invention is, for example, a water-soluble polymer that is a biological component, it is possible to melt the frozen biological sample and use it as a solution for cell administration.Furthermore, in the case of tissue or tissue-like material, it is possible to add such an aqueous polymer solution of the present invention at the organization stage and freeze-store it as it is.This is thought to reduce problems after transplantation.In such a case, the preferred water-soluble polymer for the aqueous polymer solution and the frozen solid 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.

[0118] The cryopreservation method using the aqueous polymer solution of the present invention includes the steps of containing a biological sample in the aqueous polymer solution of the present invention used for cryopreservation of the biological sample, and cooling and solidifying the aqueous polymer solution containing the biological sample.

[0119] The polymer aqueous solution of the present invention used in the cryopreservation method using the polymer aqueous solution of the present invention contains a water-soluble polymer or a salt thereof, and has an intrinsic viscosity (η) in the range of 0.20 dL / g or more and 0.95 dL / g or less. In a DSC curve during a temperature rise obtained using a differential scanning calorimeter (DSC) under the measurement conditions of the following (1) and (2), it is desirable that no endothermic peak is observed as measured by DSC, or that the temperature at which an endothermic peak is observed is higher than -1.4°C and 1.1°C or less. (Here, the DSC measurement conditions are: (1) Hold at 20°C for 1 minute, then cool to -80°C at a rate of 5°C / min, and (2) After holding at -80°C for 1 minute, the temperature is increased to 20°C at a rate of 10°C / min.) The orthogonal projection area of ​​the biological sample in the polymer aqueous solution solidified at -80°C is reduced to less than 8 / 10, preferably 1 / 3.5 or more, of the orthogonal projection area of ​​the biological sample in the culture medium (culture medium) before cooling (before freezing).

[0120] Since the polymer aqueous solution of the present invention used in the cryopreservation method using the polymer aqueous solution of the present invention has a glass transition temperature of about -23°C ± 4°C, and the vitrification is realized by the water molecules being entangled in the network of the water-soluble polymer during freezing, the cryopreservation method using the polymer aqueous solution of the present invention does not require a high cooling rate. Therefore, after immersing a biological sample in the polymer aqueous solution of the present invention, the solution is cooled to -27°C or lower, which is the glass transition temperature of the polymer aqueous solution of the present invention. For example, the polymer aqueous 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 the cells, tissues, etc. to be preserved can be stably frozen and preserved as a frozen solid while maintaining the viability.

[0121] However, the range of the cryopreservation temperature is not limited as long as it is −27° C. or lower. For example, 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.

[0122] The cryopreservation method using the aqueous polymer solution of the present invention is a slow freezing method. That is, in the freezing method using the aqueous polymer solution of the present invention, the cooling rate is preferably 10°C / min or less. More preferably, the cooling rate is about 1°C / min or less. It is believed 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.

[0123] In addition, the polymer aqueous solution of the present invention may be pH-adjusted as necessary before contacting with a biological sample. For example, when the hydrophilic group of a monomer having a hydrophilic group is a carboxylic acid group, the polymer aqueous solution containing a water-soluble polymer polymerized from such a monomer may be acidic. In addition, when a sugar has a carboxylic acid group, the polymer aqueous solution may be acidic due to such sugar. In such a case, the polymer aqueous solution may be made neutral by adjusting the pH, which may make the solution more suitable for the survival of cells to be cryopreserved, and the survival rate of the cells may be improved. There is no limitation on the salt used for adjusting the pH, and any salt commonly used for adjusting the pH of an aqueous solution may be used.

[0124] In the method for freezing and preserving a biological sample using the aqueous polymer solution of the present invention, the vitrified state is stabilized and the toxicity is low, so that the biological sample can be stably preserved for a long period of time. In this specification, the term "long-term stable preservation" means that, for example, when the aqueous polymer solution of the present invention is used, the viability of the biological sample, for example, cells, after thawing 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 preservation. In addition, in this specification, the term "long-term stable preservation" 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. In the method for freezing and preserving a biological sample using the aqueous polymer solution of the present invention, it is considered that cells can be frozen and preserved under less stressful conditions than conventional freezing and preserving methods using a DMSO solution as a freezing and preserving solution. Therefore, the cryopreservation method of the present invention can obtain a very high cell survival rate after thawing, compared with the cryopreservation method using a conventional cryopreservation liquid 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. According to the cryopreservation method using the aqueous polymer solution of the present invention, cells can be cryopreserved stably for a long period of time without changing the properties of the cells. EXAMPLES

[0125] 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".

[0126] Water-soluble polymers and aqueous polymer solutions for cryopreservation <Preparation of test samples and sample solutions> Example 1 In a pressure-resistant vessel 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 in a ratio of 20:100, and subcritical processing was performed at a processing temperature of 175°C, a processing pressure of 0.89 MPa, and a processing time of 3 minutes. The subcritically processed product was then dried by freeze drying (this drying could also be performed by the spray drying method). As a result, a water-soluble polymer was obtained, which was a mixture of polymeric hyaluronic acid with a viscosity average molecular weight of about 10,000 and hyaluronic acid with a viscosity average molecular weight of 1,000.

[0127] When this water-soluble polymer was confirmed by HPLC (Fig. 12), it was considered that the test sample of Example 1 was a mixture of high molecular weight hyaluronic acid decomposition product and low molecular weight hyaluronic acid decomposition product by subcritical treatment of hyaluronic acid, so high molecular weight hyaluronic acid was precipitated in ethanol, low molecular weight hyaluronic acid was fractionated on the supernatant side, and this fractionated supernatant was reanalyzed by HPLC.Since it was roughly consistent with the HPLC peaks (Fig. 11A-C) of hyaluronic acid of Production Examples 1-3 described later, the viscosity average molecular weight of the low molecular weight hyaluronic acid component obtained by this Example 1 was estimated to be 1000.

[0128] The intrinsic viscosity of the high molecular weight hyaluronic acid was 0.49 dL / g, and the viscosity average molecular weight was 10,000.

[0129] The test sample solution of Example 1 was obtained by dissolving 1 g of the test sample of Example 1 in 10 mL of α-MEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent is water) as a solvent (i.e., the concentration of the hyaluronic acid sample in the test sample solution is 10 w / v%). Including this Example 1, in the examples, production examples and comparative examples of the present invention, α-MEM medium is used as the solvent, but ultrapure water may be used as the solvent instead of α-MEM medium. In the DSC measurement described later, the solvent is ultrapure water instead of α-MEM. Note that each test sample obtained in the following examples was prepared to a predetermined concentration using an appropriate solvent and used in the tests for each evaluation described later.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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 sample solution of Comparative Example 1 (i.e., the DMSO concentration of the test sample in the test sample solution was 10 w / v%).

[0134] 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.

[0135] 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 sample solution of Comparative Example 3 (i.e., the gelatin concentration of the test sample in the test sample solution was 10 w / v%). The intrinsic viscosity was 0.50 dL / g.

[0136] Comparative Example 4 1g of high molecular weight hyaluronic acid (manufactured by Shanghai Easier Industrial Development Co., Ltd.) with viscosity average molecular weight of 1,000,000 is dissolved in 100mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, solvent is water) as solvent, to obtain the test sample solution of Comparative Example 4 (i.e., the concentration of the hyaluronic acid of the test sample in the test sample solution is 1w / v%). The intrinsic viscosity is 17.2dL / g.

[0137] Comparative Example 5 Hyaluronic acid (lifecore biomedical; powder) with viscosity average molecular weight of 15000 was used as the test sample. 1g of this test sample was dissolved in 10mL of αMEM medium (Gibco, product number C1257-1500BT, solvent is water) to obtain the test sample solution of Comparative Example 5 (i.e., the concentration of hyaluronic acid of the test sample in the test sample solution is 10w / v%). The intrinsic viscosity was 0.65dL / g.

[0138] Comparative Example 6 Chondroitin sulfate sodium salt A (manufactured by Sigma) with a viscosity average molecular weight of 23000 was used as a test sample. 1 g of this test sample was dissolved in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent is water) to obtain a test sample solution of Comparative Example 6 (i.e., the concentration of chondroitin sulfate of the test sample in the test sample solution was 10 w / v%). The intrinsic viscosity was 0.97 dL / g.

[0139] Comparative Example 7 The test sample of Production Example 1 (a hyaluronic acid fragment sample with a viscosity average molecular weight of 1000) was added in an amount of 1 w / v% to carboxypolylysine (viscosity average molecular weight 13400: Bioverde Co., Ltd., CryoScarless DMSO free) with 60% amino groups carboxylated, to prepare a test sample solution of Comparative Example 7. The intrinsic viscosity was 0.11 dL / g.

[0140] Comparative Example 8 The test sample used was fructan derived from shallots. The fructan was obtained by extraction and purification according to Example 2 of JP 2012-235728 A as follows.

[0141] Approximately 1 kg of dried shallots as raw material was crushed and mixed with water for extraction in which 15 g of calcium hydroxide was suspended. Extraction was performed at room temperature for 1 hour, followed by heating extraction at 95°C for 1 hour, and then heating extraction at 60°C overnight. Activated carbon was added to the extract for deodorization, and then it was filtered through diatomaceous earth to remove residue and activated carbon. The obtained extract was passed through an ultrafiltration membrane to purify fructans.

[0142] 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.

[0143] The viscosity average molecular weight of the resulting purified fructan powder was measured and found to be 12,000.

[0144] 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 8 (i.e., the test sample fructan concentration in the test sample solution was 10 w / v%). The intrinsic viscosity was 0.11 dL / g.

[0145] Comparative Example 9 Pullulan (Tokyo Chemical Industry Co., Ltd.) with a viscosity average molecular weight of 373,000 was used as a test sample. 1 g of this test sample was dissolved in 10 mL of αMEM medium (Gibco, product number C1257-1500BT, solvent is water) to obtain a test sample solution of Comparative Example 9 (i.e., the hyaluronic acid concentration of the test sample in the test sample solution is 10 w / v%). The intrinsic viscosity was 0.55 dL / g.

[0146] Example 2 The test sample of Comparative Example 5 (hyaluronic acid with viscosity average molecular weight of 15000) 1g was dissolved in αMEM medium (Gibco, product number C1257-1500BT, solvent is water) 10mL to obtain a test sample solution with a hyaluronic acid concentration of 10w / v%, and the test sample of Production Example 1 (hyaluronic acid fragment sample with viscosity average molecular weight of 1000) was added in a final concentration of 1w / v%. The pH of the obtained aqueous solution was adjusted to neutral using 10mM Tris-HCl to obtain the test sample solution of Example 2. The intrinsic viscosity was 0.65dL / g.

[0147] Example 3 The test sample of Comparative Example 9 (pullulan with viscosity average molecular weight of 373000) 1g is dissolved in αMEM medium (Gibco, product number C1257-1500BT, solvent is water) 10mL to obtain a test sample solution with a pullulan concentration of 10w / v%, and the test sample of Preparation Example 1 (hyaluronic acid fragment sample with viscosity average molecular weight of 1000) is added to the test sample solution with a final concentration of 1w / v% to obtain the test sample solution of Example 3. The intrinsic viscosity is 0.55dL / g.

[0148] Example 4 The test sample of Comparative Example 9 (pullulan with a viscosity average molecular weight of 373,000) was dissolved in 10 mL of αMEM medium (Gibco, product number C1257-1500BT, solvent is water) to obtain a test sample solution with a pullulan concentration of 10 w / v%, to which the test sample of Preparation Example 1 (hyaluronic acid fragment sample with a viscosity average molecular weight of 1000) was added at a final concentration of 5 w / v%. The pH of the obtained aqueous solution was adjusted to neutral using 10 mM Tris-HCl to obtain the test sample solution of Example 4. The intrinsic viscosity was 0.55 dL / g.

[0149] Example 5 In a pressure-resistant vessel 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 in a ratio of 20:100, and subcritical processing was carried out at a processing temperature of 175°C, a processing pressure of 0.89MPa, and a processing time of 3.5 minutes. The subcritically processed product was then dried by freeze-drying or spray-drying. As a result, a hyaluronic acid sample with a viscosity average molecular weight of approximately 6000 was obtained. 1g of this hyaluronic acid 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 sucrose (Nacalai Tesque, molecular weight (simulated to be the same as viscosity average molecular weight) is 342) and glucuronic acid (Fujifilm Wako Pure Chemical, molecular weight (simulated to be the viscosity average molecular weight) is 194) are added at a final concentration of 1w / v% to obtain a test cryopreservation solution of Example 5. The intrinsic viscosity was 0.32dL / g.

[0150] Example 6 1g of the test sample (hyaluronic acid with viscosity average molecular weight of 15000) of Comparative Example 5 is dissolved in 10mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, solvent is water) to obtain an aqueous solution with a hyaluronic acid concentration of 10w / v%, and the test sample (hyaluronic acid fragment sample with viscosity average molecular weight of 2000) of Preparation Example 2 is added in a final concentration of 1w / v% to obtain the test sample solution of Example 6. The intrinsic viscosity is 0.47dL / g.

[0151] <Test Example 1: Cryopreservation of primary human mesenchymal stem cells using a test sample 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 antibodies were suspended in the test sample solutions (serum-free) of Examples 1 and 5 and Comparative Examples 1, 2, 4, 5, 7 and 8 at a concentration of 100000000000 / mL.

[0152] Thereafter, the cell suspension containing each test sample solution was frozen in a slow cell freezer (Nalgene (registered trademark) Mr. Frosty) in a -80°C freezer to obtain a frozen solid. The frozen cell suspension containing each test sample was stored at -80°C for 7 days and then rapidly thawed in a 37°C warm bath. The cell suspension containing each test sample solution after thawing was evaluated for cell viability by trypan blue staining immediately after thawing. The results are shown in Figure 1 and Table 1 (the results of Example 5 are only in Table 1).

[0153] Fig. 1 shows the cell viability after thawing of cells cryopreserved in a medium without cryopreservation solution, i.e., in the test sample solution of Comparative Example 2, or in the test sample solutions of Example 1 and Comparative Examples 1, 4, 5, 7 and 8. As shown in Fig. 1, in Example 1, i.e., the hyaluronic acid sample obtained by subcritical treatment of hyaluronic acid has a viscosity average molecular weight of about 10,000, and further contains a hyaluronic acid cleavage product having a viscosity average molecular weight of 1000, i.e., a molecular weight smaller than 10,000, is a water-soluble polymer in a polymer aqueous solution, has an intrinsic viscosity of 0.49 dL / g, and generates 0 J / g in DSC measurement during the temperature drop process (see Test Example 2 below). In the test sample solution of Example 1, a remarkably high preservation effect of more than 90% was obtained.

[0154] In the frozen solid obtained by freezing the test sample of Example 1, no endothermic peak was observed in the DSC measurement during the heating process (see Test Example 2 below). Therefore, it is presumed that the water molecules do not form ice crystals, but are free water or bound water adsorbed or entangled by hyaluronic acid molecular chains.

[0155] In addition, the brightness difference between the solvent matrix region and the cell region of this frozen solid is as small as 2 (see Test Example 8 below), so it is considered that the state of water molecules in the solvent matrix region and the cell region is similar, and ice crystals are not formed inside the cells. Furthermore, since the orthogonal projection area of ​​the cells when the test sample solution is solidified at -80°C is shrunk to 38% of the orthogonal projection area of ​​the cells in the culture solution (culture medium) before solidification (see Test Example 9 below), it is considered that hyaluronic acid does not enter the cells, and water molecules move outside the cells due to the osmotic pressure difference. It is considered that the water molecules that move outside the cells are replaced with low molecular weight hyaluronic acid or high molecular weight hyaluronic acid, and do not cause problems such as destruction of cell tissue by remaining around the cells and forming ice crystals, and hyaluronic acid does not enter the cells and harm the cells. Therefore, unlike DMSO in Comparative Example 1, it has low cytotoxicity and does not form ice crystals inside or around the cells, so it is also excellent in the cryopreservation effect of cells.

[0156] The preservation effect of this test sample solution was even higher than that of Comparative Example 1, which contained DMSO, a commonly used cryopreservative, as a cryopreservative. It can be seen that the aqueous polymer solution of the present invention is an excellent cryopreservative solution with low cytotoxicity.

[0157] As can be seen from Table 1, comparing Examples 3 and 4 with Comparative Example 9, when the heat release amount measured by DSC during the temperature drop of the aqueous polymer solution exceeds 65% of that of standard water (ultrapure water), and when the heat absorption amount measured by DSC during the temperature rise of the frozen solidified body exceeds 65% of that of standard water (ultrapure water), the cell viability decreases even if the conditions such as the limiting viscosity and cell shrinkage rate are met.

[0158] The test sample solution of Comparative Example 4 contained high molecular weight hyaluronic acid with a molecular weight of 1,000,000 as the test sample, but although the intrinsic viscosity was high at 17.2 dL / g and no endothermic peak was confirmed (see Test Example 2 below), almost no cell protective effect was observed.

[0159] In the frozen solidified product of Comparative Example 4, no endothermic peak was observed in the DSC measurement during the temperature rise process, but the frozen solidified cells did not shrink, and therefore it is believed that water molecules remain within the cells. In addition, the brightness difference between the solvent matrix region and the cell region of the frozen solidified product was large at 4 (see Test Example 8 below), and the cell region was dark, and it is believed that the water molecules within the cells formed ice crystals. For this reason, it is presumed that the ice crystals destroyed the cells, and the cell preservation effect was not observed.

[0160] In Comparative Example 2, which did not contain a cryopreservative, the cell viability was approximately 0%.

[0161] In addition, in Comparative Example 7, which contains carboxypolylysine as a test sample and has a low intrinsic viscosity of 0.11 dL / g, the cell protection effect was not as good as that of Example 1 or Comparative Example 1, which uses DMSO as a cryoprotectant. In the test sample solution of Comparative Example 7, the intrinsic viscosity of polylysine is low, so that the water molecules that have moved outside the cells and the polymeric or low molecular weight sugars around the cells are diffused, and it is presumed that the replacement of the water molecules with the polymeric or low molecular weight sugars does not proceed, and the water molecules remain around the cells to form ice crystals. In addition, since the area of ​​the cells after freezing is larger than the area in the culture solution (in the medium) (see Test Example 9 below), it is considered that polylysine has penetrated into the cells or the cells have expanded due to the ice crystals. It is presumed that the cells are damaged and sufficient preservation effect cannot be obtained.

[0162] Furthermore, Comparative Example 8, which used fructan, also had a small intrinsic viscosity of 0.11 dL / g, and it is thought that the water molecules that moved outside the cells were not replaced by high molecular weight or low molecular weight sugars around the cells, and that approximately half of the cells burst and died due to the formation of ice crystals.

[0163] Furthermore, it can be seen from Example 5 that glucuronic acid and sucrose also have the function of adjusting the endothermic peak to around 0.7°C (the endothermic peak of hyaluronic acid with a viscosity average molecular weight of approximately 6000 is -1.4°C).

[0164] [Table 1]

[0165] In addition, since the polymer aqueous solution of the present invention showed a high cell preservation effect even when prepared using a culture medium such as αMEM medium instead of water as a solvent, the polymer aqueous solution of the present invention can be added directly to a cell culture medium to suspend the cells, and then frozen for preservation. It is believed that it is possible to achieve a higher cell survival rate and maintain the properties of the cells without the need for centrifugation of the cells to be preserved.

[0166] <Test Example 2: Analysis of each test sample solution by differential scanning calorimeter> The solvent of each test sample solution in Examples 1 to 6, Comparative Examples 1 to 9, and Production Examples 1 to 3 was changed from αMEM medium to ultrapure water to prepare 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.

[0167] The DSC curves during the temperature rise obtained under the above DSC measurement conditions for Example 1, Comparative Example 5, and ultrapure water (Comparative Example 2) are shown in Figure 2A. Figure 2B is an enlarged view of Figure 2A in the vicinity of the glass transition temperature of the aqueous polymer solution of the present invention. The DSC curves for Example 1, Comparative Example 1, and ultrapure water (Comparative Example 2) are shown in Figures 3A to 3C.

[0168] In addition, the temperature at which an endothermic peak estimated to be due to the melting of water was observed in the DSC curve during the temperature rise process of each test sample solution, the endothermic amount of the endothermic peak (amount of heat absorbed during temperature rise), and the heat release amount of an exothermic peak observed in the DSC curve during the temperature drop process (amount of heat released during temperature drop) were analyzed. The results are shown in Table 1. In Table 1, the endothermic amount of the endothermic peak is shown as a percentage (%) when the heat release amount of 112.4 J / g measured with ultrapure water (Comparative Example 2; used as the reference solution) is taken as 100. In Table 1, the heat release amount of the exothermic peak is shown as a percentage (%) when the heat release amount of 106 J / g measured with ultrapure water (Comparative Example 2; used as the reference solution) is taken as 100.

[0169] As shown in Figures 2A and 2B, in Comparative Example 2 in which the test sample was ultrapure water (i.e., a sample in which the test sample was ultrapure water), only the ice crystal melting peak of free water was observed (Figure 2A). In Comparative Example 5 in which the test sample was hyaluronic acid with a viscosity average molecular weight of 15,000, a shift in the ice crystal melting peak and a drop in the freezing point were observed compared to Comparative Example 2 (Figure 2A), and a glass transition was confirmed near -20°C (Figure 2B). On the other hand, in the test sample of Example 1, which contains hyaluronic acid with a viscosity average molecular weight of about 10,000 as well as a scission product of hyaluronic acid (low molecular weight hyaluronic acid with a viscosity average molecular weight of 1000), the ice crystal melting peak was extremely small and almost not observed (Figure 2A), and only a glass transition was confirmed near -23°C ± 4°C (Figure 2B). This result indicates that in the aqueous polymer solution of Example 1, water exists in the matrix in an unfrozen state (free water) or in a state entrapped by the polymer (bound water), and that recrystallization of water upon melting is suppressed, which is a significantly advantageous effect of the aqueous polymer solution of the present invention.

[0170] Furthermore, as shown in FIG. 3A, in Comparative Example 2 in which the test sample was ultrapure water, a very large heat of heat associated with ice crystal formation was observed during the temperature drop process. Also, a large amount of heat of fusion associated with ice crystal melting was observed during the temperature rise process. In Comparative Example 1 in which the test sample was DMSO, as shown in FIG. 3B, only a small peak associated with ice crystal formation was observed, 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 polymer aqueous solution of the test sample of Example 1 (hyaluronic acid with a viscosity average molecular weight of 10,000 containing a cleavage product of hyaluronic acid), as shown in FIG. 3C, almost no melting peak was observed, and the peak associated with ice crystal formation was much smaller than the peak observed in Comparative Example 1. Therefore, it is considered that the polymer aqueous solution containing the test sample of Example 1 hardly forms ice crystals, and is in an extremely good vitrified state.

[0171] In addition, the temperature at which endothermic peak is observed (endothermic peak temperature) is -1.4°C in Comparative Example 5 (test sample is hyaluronic acid with viscosity average molecular weight of 15000), but -0.4°C in Example 2, which is made by mixing Comparative Example 5 with hyaluronic acid fragments of Preparation Example 1 with viscosity average molecular weight of 1000 or less, and is close to the endothermic peak appearance temperature of pure water, 0.7°C. The endothermic peak temperature of Preparation Example 1 is -8.1°C, and if it is added to a water-soluble polymer with a lower endothermic peak appearance temperature than pure water, it is expected that the endothermic peak appearance temperature will be further reduced, but conversely, the endothermic peak appearance temperature will rise and be close to the endothermic peak appearance temperature of pure water, or it can be adjusted so that endothermic peak or exothermic peak does not appear.

[0172] On the other hand, the endothermic peak appearance temperature of Comparative Example 9 (the test sample was pullulan with a viscosity average molecular weight of 373,000) was 1.49° C., which was higher than the endothermic peak appearance temperature of pure water, 0.7° C. When this Comparative Example 9 was mixed with Production Example 1 (endothermic peak appearance temperature: −8.1° C.) (Examples 3 and 4), the endothermic peak appearance temperature was not increased but decreased to 0.83° C., which is close to the endothermic peak appearance temperature of pure water, 0.7° C.

[0173] In other words, it was revealed that when hyaluronic acid with a molecular weight of 1000 or less is mixed with a water-soluble polymer whose endothermic peak appears at a lower temperature than pure water, the peak appears temperature of the polymer aqueous solution rises and is closer to that of pure water, and when hyaluronic acid with a molecular weight of about 1000 is mixed with a water-soluble polymer whose endothermic peak appears at a higher temperature than pure water, the peak appears temperature of the polymer aqueous solution falls and is closer to that of pure water.

[0174] In contrast, the endothermic peak temperature of the hyaluronic acid fragments having a viscosity average molecular weight of 2000 or less in Production Example 2 was -0.3°C, as shown in Table 1. The endothermic peak temperature of Example 6, in which the low molecular weight sample of Production Example 2 was mixed with Comparative Example 5 (endothermic peak temperature of -1.4°C), was lowered to -1.6°C.

[0175] This result shows that by using a low molecular weight saccharide or its salt, particularly preferably a low molecular weight saccharide having a viscosity average molecular weight of 3000 or less, the endothermic peak temperature of the polymer aqueous solution can be adjusted to be around the endothermic peak temperature of pure water, 0.7° C. When sucrose, trehalose, or glucuronic acid was used as the low molecular weight saccharide or its salt having a viscosity average molecular weight of 3000 or less, the same effect as that of the sample in Production Example 1 on the endothermic peak temperature was observed.

[0176] In addition, the heat release amount of the exothermic peak during the temperature drop process of the test sample solutions of Examples 1 to 6 was 65% or less, which was smaller than the corresponding heat release amount of the reference solution made of water (Comparative Example 2). In addition, the heat release amount of the endothermic peak during the temperature rise process of the test sample solutions of Examples 1 to 6 was 65% or less, which was smaller than the corresponding heat release amount of the reference solution made of water (Comparative Example 2). It can be seen that in the aqueous polymer solutions of the present invention, water is not crystallized but is solidified in a glassy state.

[0177] The endothermic peak temperatures of the test sample solutions in Examples 1 to 6 are near the endothermic peak temperature of water, 0.7°C, i.e., they behave similarly to pure water, and the heat release value of the exothermic peak is smaller than that of the exothermic peak observed during the cooling process of water, which indicates that bound water and free water that do not form ice crystals remain in the frozen aqueous polymer solution of the present invention. It is believed that the water-soluble polymer of the present invention achieves vitrification by trapping water molecules in its matrix when frozen.

[0178] <Test Example 3: Evaluation of long-term preservation effect in cryopreservation using a test sample solution 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 sample solutions (serum-free) of Example 1 and Comparative Example 1 at a concentration of 1000 / mL. The cell suspensions containing each test sample solution were then frozen in a slow cell freezer (Nalgene (registered trademark) Mr. Frosty) in a -80°C freezer to obtain frozen solids. The frozen cell suspensions containing each test sample solution were stored at -80°C for up to 12 months, and then the cell suspensions containing each test sample solution were taken out at the designated storage period and quickly thawed in a 37°C warm bath. The cell viability of the cell suspensions containing each test sample 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.

[0179] From the results shown in FIG. 4A, it can be seen that in the case of cryopreservation using the aqueous polymer solution of the present invention, the frozen cells show a high cell viability of 95% or more, which remains almost unchanged even after 5 months of storage. On the other hand, in the case of cryopreservation using the sample 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 of Comparative Example 1 is below 50%. This result is in contrast to the aqueous polymer solution of the present invention, in which high cell viability is still maintained even after 3 months. After 6 months of cryopreservation, the cell viability decreased by less than 10% in the case of cryopreservation using the aqueous polymer solution of the present invention, while the cell viability decreased to about 25% in the case of cryopreservation using the sample solution of Comparative Example 1. Furthermore, in the case of cryopreservation using the aqueous polymer solution of the present invention, the cell viability was still high even after 12 months of cryopreservation, and the decrease in cell viability was only less than 15%.

[0180] Therefore, it is evident that the aqueous polymer solution of the present invention enables cells to be cryopreserved stably for a long period of time with a high cell survival rate.

[0181] In addition, when cells stored for a long period at -80°C 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 when the aqueous polymer solution of the present invention was used, whereas it decreased to about 60% in Comparative Example 1. The cells cryopreserved using the aqueous polymer solution of the present invention still had a cell viability of 40% or more even after one week of storage at 4°C. This indicates that the cells that survived after thawing in the aqueous polymer solution of the present invention were essentially cells that were normally proliferating, and that the aqueous polymer solution of the present invention did not damage the cells during storage, confirming that the aqueous polymer solution of the present invention has a very excellent cell preservation effect.

[0182] <Test Example 4: Evaluation of properties after cryopreservation using a test sample solution 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 sample solutions (serum-free) of Example 1 and Comparative Example 1 at a concentration of 100 x 10 cells / mL. The cell suspensions containing each test sample solution were then frozen in a slow cell freezer (Nalgene (registered trademark) Mr. Frosty) in a -80°C freezer to produce frozen solids. The frozen cell suspensions containing each test sample solution were stored frozen at -80°C for six months. After six months, the cell suspensions containing each test sample solution were rapidly thawed in a 37°C warm bath, washed with αMEM, and then plated in a 6-well plate with 2.5 x 10 cells. 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.

[0183] As shown in Fig. 5A, in the case of cryopreservation in the presence of the sample solution of Comparative Example 1 containing DMSO as a cryopreservative, the HGF production in cells after thawing was high. On the other hand, in the case of cryopreservation using the test sample solution of Example 1 of the present invention, which is hyaluronic acid with a viscosity average molecular weight of 10000 containing hyaluronic acid cleavage products, 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 preserved in the presence of DMSO. Since such high HGF production was not observed in the case of cell preservation using the polymer aqueous solution of the present invention, it can be seen that cells are stably protected in the polymer aqueous solution of the present invention.

[0184] 5B, the amount of IL-10 produced was high in the cells cryopreserved using the test sample solution of Example 1, and was very low in the cells cryopreserved using the test sample solution of Comparative Example 1. This result shows that the aqueous polymer solution of the present invention can satisfactorily cryopreserve cells while maintaining their functions.

[0185] The aqueous polymer solution of the present invention does not contain cytotoxic compounds such as DMSO and ethylene glycol. Therefore, unlike conventional cryopreservation solutions, the aqueous polymer solution of the present invention has the remarkable effect of enabling biological samples to be cryopreserved while maintaining their properties without exposing them to stressful conditions.

[0186] In addition, because serum or serum-derived proteins are not added, there is no contamination with bacteria or viruses. However, it is possible to add proteins that are not contaminated by bacteria or viruses. In addition, it is possible to use compounds that are cell-permeable and toxic at low concentrations that do not impair cell function.

[0187] <Test Example 5: Evaluation of properties (undifferentiated state) after cryopreservation using a test sample solution 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 sample solutions (serum-free) of Example 1 and Comparative Example 1 at a concentration of 1000 / mL. The cell suspensions containing each test sample solution were then frozen in a slow cell freezer (Nalgene (registered trademark) Mr. Frosty) in a -80°C freezer to obtain frozen solids. The frozen cell suspensions containing each test sample solution were frozen and stored at -80°C for 6 months. After 6 months, the cell suspensions containing each test sample solution were removed and quickly thawed in a 37°C warm bath. The expression intensities of CD90, CD44, and CD105 in the thawed cell suspensions containing each test sample solution were analyzed by flow cytometry. The results are shown in Figure 6.

[0188] CD90, CD44, and CD105 are typical surface proteins expressed in undifferentiated mesenchymal stem cells, 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 in the aqueous polymer solution of Example 1 than in cells cryopreserved in the test sample solution of Comparative Example 1. Therefore, it can be seen that the cells cryopreserved in the aqueous polymer solution of Example 1 maintain the expression of undifferentiated markers. That is, the cells cryopreserved in the aqueous polymer solution of Example 1 can maintain an undifferentiated state. It can be seen that the cryopreservation using the aqueous polymer solution of the present invention can reduce the influence on the differentiated state seen in the case of preservation in the test solution of Comparative Example 1.

[0189] These results demonstrate that the cryopreservation method using the aqueous polymer 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.

[0190] <Test Example 6: Evaluation of aqueous polymer solutions containing various samples as frozen storage solutions> Cultured primary canine mesenchymal stem cells (cyagen C160) were added at 1 × 10 6 The cells were suspended in the test sample solutions (serum-free) of Examples 1 to 4 and 6 and Comparative Examples 3, 6 and 9 at a concentration of 10 ...

[0191] From the results of cell viability shown in Table 1 together with the results of Test Example 1, it can be seen that the water-soluble polymer in the present invention is not limited to hyaluronic acid, and even if it is another water-soluble polymer, it will show high cell protection effect as long as the polymer aqueous solution has a certain limiting viscosity and endothermic peak temperature.In addition, from the results of Comparative Example 9 and Examples 3 and 4 (Comparative Example 9 with Production Example 1 added), it was found that the water-soluble polymer is used in combination with a sugar or its salt having a smaller molecular weight, and the polymer aqueous solution shows good cell protection effect.As shown in the results of Test Example 2, it is presumed that this is because the endothermic peak temperature of the sample solution can be adjusted to the endothermic peak temperature of pure water, 0.7 ° C, by adding a low molecular sugar or its salt to the polymer aqueous solution. Example 6 is a test sample solution obtained by adding low molecular weight saccharides (hyaluronic acid fragment sample with viscosity average molecular weight of 2000) of Production Example 2 to a polymer aqueous solution of hyaluronic acid with viscosity average molecular weight of 15000 (Comparative Example 5). As shown in the results of Test Example 2, the endothermic peak temperature of the DSC curve is -1.6°C, which is lower than the endothermic peak temperature (-1.4°C) of Comparative Example 5, and is far from the endothermic peak appearance temperature of pure water, 0.7°C. Also, in this Test Example 6, Example 6 did not obtain a cell viability as high as other Examples. From this result, it was found that it is important to adjust the endothermic peak temperature of the test sample solution to a desired range by mixing low molecular weight saccharides or their salts into the polymer aqueous solution, and this can obtain a high cell viability.

[0192] In addition, when the test sample solution prepared by adding saccharides or their salts was acidic, a good cell protection effect was obtained by neutralizing the test sample solution by pH adjustment. It is considered that the pH adjustment provides more suitable conditions for cell survival. In order for the added low molecular weight saccharides or their salts to act as cell protective components and further improve the cell survival rate of cells after cryopreservation, it was necessary to add low molecular weight saccharides or their salts to the polymer aqueous solution in an amount of 1 to 5 w / v% relative to the water-soluble polymer. On the other hand, even if the amount of the added low molecular weight saccharides or their salts was more than 10 w / v% relative to the water-soluble polymer, no further effect on the improvement of cell survival rate was observed.

[0193] From Table 1, it can be seen that when the intrinsic viscosity (η) of the test sample solution is outside the range of 0.20 dL / g or more and 0.95 dL / g or less, or when the endothermic peak temperature of the DSC curve is -1.4°C or less and higher than 1.1°C, the cell viability after cryopreservation decreases. As is clear from Table 1, even if only one of the intrinsic viscosity and the endothermic peak temperature is within the specified range, the desired effect (high cell viability) cannot be obtained. It is considered that the intrinsic viscosity (η) within the above-mentioned specified range allows the aqueous polymer solution to help the discharge of water molecules from within the biological sample, and to efficiently replace the discharged water molecules with the polymer and / or low molecular weight sugars around the biological sample. Furthermore, it is considered that the endothermic peak temperature is higher than -1.4°C and about 1.1°C or less, which indicates that the aqueous polymer solution can retain water molecules within the polymer matrix when frozen.

[0194] Furthermore, the results of Test Example 6 demonstrated that the aqueous polymer solution of the present invention exhibited a favorable cryoprotective effect not only on human but also on canine mesenchymal stem cells.

[0195] <Test Example 7: Evaluation of cells in frozen test sample solution (evaluation of intracellular vitrification state)> Cultured primary canine mesenchymal stem cells (cyagen C160) were added at 1 × 10 6The cells were suspended in the test sample solutions (serum-free) of Example 1 and Comparative Example 1 at a concentration of 1000000000000 / mL. As a control, a control suspension was prepared in the test sample solution of Comparative Example 2 consisting of αMEM medium without a cryopreservative. Then, 5 μL of the cell suspension and the control suspension containing each test sample 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. 7A to 7C.

[0196] FIG. 7A 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. 7B shows the results for Comparative Example 1, in which DMSO is used as the test sample. It can be seen that the cells also turn dark in FIG. 7B, and that minute ice crystals have formed. FIG. 7C shows the results for the aqueous polymer solution containing the test sample of Example 1. It can be seen that the cells turn light, and that the inside of the cells has been vitrified into an amorphous state.

[0197] From this result, it can be seen that the polymer aqueous solution of the present invention freezes the inside of cells in a vitrified state.And it can be seen that the vitrified state is formed more stably by including sugars that are the cleavage products of hyaluronic acid.It is considered that vitrification occurs stably and efficiently because the formation of ice crystals around cells is suppressed by sugars with low molecular weight.

[0198] <Test Example 8: Evaluation of intracellular vitrification state using brightness difference> Cultured primary canine mesenchymal stem cells (cyagen C160) were added at 1 × 10 6The cells were suspended in the test sample solutions (serum-free) of Examples 1 to 6 and Comparative Examples 1 to 9 at a concentration of 10000000000 / mL. As a control, a control suspension was prepared 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 sample 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 of Example 1 and Comparative Examples 1 and 2 are shown in FIGS. 8A and 8B.

[0199] The difference (absolute value) in brightness between the inside and outside of the cell in the observation image shown in Figure 8A was analyzed using the image analysis software ImageJ. 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, and 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.

[0200] As a result of the measurement, 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. 8B). Similarly, for Examples 2 to 6 and Comparative Examples 3 to 9, the Munsell value of the solvent region and the Munsell value of the intracellular region were obtained, and the value difference was calculated. The results are shown in Table 1.

[0201] When ice crystals are formed during freezing, visible light is blocked and the brightness decreases. Therefore, it is preferable that the Munsell brightness is 2 or more in the solvent region and the intracellular region. Munsell brightness of this level indicates that ice crystals are not formed in the solvent region and the intracellular region, that is, the cells are well vitrified. In Example 1, the Munsell brightness of the solvent region was 4, the frozen cells were bright, and the Munsell value of the intracellular region was 2. Therefore, it was found that ice crystals were not formed in either region.

[0202] In addition, the difference in brightness between the Munsell brightness of the solvent region and the Munsell value of the intracellular region was 2 in Examples 2 to 6, as in Example 1. That is, in the Examples, the brightness of the intracellular region and the brightness of the solvent region are close to each other. This result shows that in the cryopreservation using the polymer aqueous solution containing the test sample of the Examples, the frozen state is almost the same in the matrix region and the intracellular region, that is, ice crystals are not formed in the region within the biological sample. It is desirable that the difference in brightness 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.

[0203] <Test Example 9: Evaluation of cells in frozen sample solution> Cultured primary canine mesenchymal stem cells (cyagen C160) were added at 1 × 10 6The cells were suspended in the test sample solutions (serum-free) of Examples 1 to 6, Comparative Examples 1 to 9, and Production Examples 1 to 3 at a concentration of 1000 / mL. Then, 5 μL of the cell suspension containing each test sample solution was 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) to solidify, and observed with a transmitted light microscope (Olympus BX53). Images were taken from above the normal direction of the cooling stage before cooling (before solidification) and after cooling to -80°C (solidified state). The cell area of ​​each cell was calculated (orthogonal projection area of ​​the cell) using image analysis software ImageJ in the images taken before and after cooling, and the change in the orthogonal projection area of ​​the cell due to solidification was determined. The ratio of the cell area after solidification to the cell area before solidification was calculated and used as the cell shrinkage rate (%). The results are shown in Table 1.

[0204] The cells frozen in the test sample solutions of Examples 1 to 6 showed cell shrinkage rates of 38%, 33%, 67%, 76%, 40% and 50%, respectively. In cryopreservation using the test sample solutions of Examples 1 to 6, the discharge of water from the cells to be frozen is promoted, and the cells are well dehydrated during freezing. In freezing the biological samples using the aqueous polymer solutions of Examples 1 to 6, the water-soluble polymer and / or low molecular weight saccharides of the present invention are present in sufficient proximity to the cells in all cases, and the water molecules that move from inside the cells to outside the cells due to the osmotic pressure difference between the intracellular fluid and the aqueous polymer solution are trapped in the matrix of the water-soluble polymer, further promoting the discharge of water from inside the cells, and the increased concentration of proteins and saccharides in the cells makes the water in the cells supercooled, and the vapor pressure difference between the supercooled water further promotes the movement of water molecules through the cell membrane to the outside of the cells, thereby preventing or suppressing freezing in the cells and the growth of ice crystals. In addition, since the cells are shrinking, it can be seen that the water-soluble polymers and the like do not penetrate into the cells.

[0205] In contrast, in Comparative Example 1, in which DMSO was used as the test sample, and Comparative Example 2, in which only the medium was used, the orthogonal projection area of ​​the cells increased after freezing. In addition, in the test sample solutions of Comparative Examples 3, 4, and 6, in which the intrinsic viscosity (η) was outside the range of 0.20 dL / g or more and 0.95 dL / g or less, the cells had almost the same orthogonal projection area as before freezing. Furthermore, in the test sample solutions (Comparative Examples 5 and 9) in which the intrinsic viscosity (η) was within the range of 0.20 dL / g or more and 0.95 dL / g or less, the endothermic peak temperature of the DSC curve was outside the endothermic peak temperature of water, 0.7°C, and the temperature was below -1.4°C and above 1.1°C, sufficient cell shrinkage was not observed. In the test sample solutions of these comparative examples, dehydration of the cells did not occur or was insufficient, and therefore the survival rate of the cells after freezing was also considered to be lower than that of Examples 1 to 6 in which the cells were well dehydrated. In Comparative Examples 7 and 8, the orthogonal projection area of ​​the cells was increased from that before freezing. In Comparative Example 8, cell rupture due to freezing was observed. In Comparative Examples 7 and 8, it is believed that the water-soluble polymer penetrated into the cells. Furthermore, the cell viability after cryopreservation in Comparative Examples 7 and 8 was also low. Such cell-permeable water-soluble polymers expand and damage the cells when frozen, and therefore are considered to be unsuitable as water-soluble polymers for polymer aqueous solutions used for cryopreservation of biological samples. Furthermore, in Production Examples 1 to 3, which have a small intrinsic viscosity (η), no change was observed in the orthogonal projection area of ​​the cells, and it was presumed that dehydration of the cells did not occur.

[0206] In cryopreservation using the aqueous polymer solution of the present invention, the inside of the cells is effectively dehydrated during freezing, so that damage to the cells during freezing is significantly reduced, and it is believed that this results in a high survival rate of the thawed cells.

[0207] <Test Example 10: 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 6The cells were suspended in the test sample solutions (serum-free) of Example 1 and Comparative Example 1 at a concentration of 10 ...

[0208] 9, when cells were cryopreserved using the aqueous polymer solution of the present invention, a high cell viability was obtained for all cells, which 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 aqueous polymer 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 origin of the cells.

[0209] <Test Example 11: Evaluation of the effect of low molecular weight sugars on cell viability> Cultured primary canine mesenchymal stem cells (cyagen C160) were added at 1 × 10 6 The cells were suspended in each test sample solution (serum-free) of Production Examples 1 to 3 at a concentration of 10 ...

[0210] As shown in Table 1, the intrinsic viscosity (η) of low molecular weight saccharides or their salts (viscosity average molecular weight is 1000, 2000, 3000, respectively) that are smaller than the molecular weight of the water-soluble polymer of the present invention is 0.08, 0.14, 0.19, respectively, which is outside the desired range of 0.20 dL / g or more and 0.95 dL / g or less. This Test Example 11 has revealed that such low molecular weight saccharides or their salts alone cannot achieve cell survival effect. In Production Example 3, a cell survival rate of about 10% was observed, which is presumably due to the hyaluronic acid with a molecular weight larger than 3000 that may be contained in the test sample of Production Example 3.

[0211] <Test Example 12: 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), and the components of each test sample were analyzed by HPLC. As the mobile phase, a 16 mM NaH2PO4 aqueous solution was used as solution A, and an 800 mM NaH2PO4 aqueous solution was used as solution B. The components were separated using a ZORBAX NH2 (Agilent Technologies, Inc., column size φ4.6×250 mm, particle size 5 μm) normal phase HPLC column at a flow rate of 1.0 mL / min, a column temperature of 40° C., and a detection wavelength of 210 nm. The gradient conditions were mobile phase B concentration 0% (0 min) → mobile phase B concentration 100% (60 min). The results are shown in Figures 11A to C. In addition, as standards, aqueous solutions containing 0.2 wt% of hyaluronic acid oligosaccharides, HA02 (disaccharide), HA04 (tetrasaccharide), HA06 (hexasaccharide), HA08 (octasaccharide), and HA10 (decasaccharide) (all manufactured by Iduron Co., Ltd.), were prepared and analyzed by HPLC in the same manner. The results are shown in Figure 11D.

[0212] 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 11D. Figure 11A 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.

[0213] <Test Example 13: 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 processing) 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.

[0214] Similarly, when compared with Fig. 11D, it can be seen that 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.

[0215] From the above results, it can be seen that the aqueous polymer solution and the frozen solidified product of the present invention have a remarkable effect of stably vitrifying the inside of a biological sample and preventing or suppressing the growth of ice crystals in the biological sample, thereby enabling cryopreservation of the biological sample with high cell viability, basically without the need for the addition of cell-permeable and cytotoxic compounds such as DMSO and ethylene glycol, and / or serum or serum-derived proteins, etc. Cells are well protected and their properties are maintained.

[0216] It is possible to add proteins that are free of bacterial or viral contamination, and it is also possible to use compounds that are cell-permeable and toxic at low concentrations that do not impair cell function.

[0217] In addition, the frozen solidified material of the present invention has the effect of making it possible to determine whether or not the cells have been cryopreserved satisfactorily, without thawing, by DSC measurement, brightness measurement, and measurement of the orthogonal projected area of ​​the cells.

Claims

1. A polymer aqueous solution containing a water-soluble polymer or a salt thereof in an aqueous solvent and used for cryopreservation of a biological sample, comprising: The intrinsic viscosity (η) of the aqueous polymer solution is 0.20 dL / g or more and 0.95 dL / g or less; the orthogonal projection area of ​​the biological sample in a state in which the aqueous polymer solution is solidified at −80° C. is reduced to less than 8 / 10 of the orthogonal projection area of ​​the biological sample before solidification; The polymer aqueous solution is characterized in that, in a DSC curve during a temperature drop process obtained using a differential scanning calorimeter (DSC) under the measurement conditions of the following (1) to (2), the heat generation amount of an exothermic peak during the temperature drop process is 0 J / g or 65% or less of the corresponding heat generation amount of a reference solution consisting of water. (DSC measurement conditions) (1) After holding at 20°C for 1 minute, the temperature was decreased to -80°C at a rate of 5°C / min. (2) After holding at -80°C for 1 minute, the temperature was increased to 20°C at a rate of 10°C / min.

2. 2. The polymer aqueous solution according to claim 1, wherein the orthogonal projection area of ​​the biological sample in a state where the polymer aqueous solution is solidified at -80°C is reduced to 1 / 3.5 or more of the orthogonal projection area of ​​the biological sample before solidification.

3. 3. The aqueous polymer solution according to claim 1, wherein in a DSC curve during a temperature rise obtained under the measurement conditions (1) to (2), no endothermic peak is observed during the temperature rise, or the endothermic peak is greater than −1.4° C. and equal to or less than 1.1° C.

4. 4. The aqueous polymer solution according to claim 1, wherein the aqueous polymer solution has a calorific value of an exothermic peak measured by DSC of 0 J / g or 40% or less of the corresponding calorific value of a reference solution consisting of water.

5. 5. The polymer aqueous solution according to claim 1, wherein the water-soluble polymer or its salt is present in an amount of 0.1 w / v % or more and 20 w / v % or less.

6. The polymer aqueous solution according to any one of claims 1 to 5, wherein the water-soluble polymer is a water-soluble polymer containing a sugar residue.

7. The aqueous polymer solution according to any one of claims 1 to 6, wherein the biological sample is a cell, a tissue, or a tissue-like substance such as a membrane or an aggregate.

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

9. The polymer aqueous solution according to claim 7, wherein the biological sample is a sperm, an egg, or a fertilized egg.

10. The polymer aqueous solution according to any one of claims 1 to 9, which is used for vitrification preservation of the biological sample.

11. A method for preventing or inhibiting ice crystal growth in a biological sample during cryopreservation, comprising contacting the biological sample with an aqueous polymer solution according to any one of claims 1 to 10.

12. A solidified body comprising a matrix containing an aqueous solvent and a water-soluble polymer or a salt thereof, and a biological sample, The biological sample has an orthogonal projection area that is reduced to less than 8 / 10 of that before solidification, a difference between a lightness in the Munsell color system of a region occupied by the biological sample and a lightness in the Munsell color system of a region occupied by the matrix is ​​3 or less when viewed from a light exit side when visible light is transmitted through the region; A solidified body, characterized in that in a DSC curve during a temperature rise obtained under the measurement conditions of (1) to (2), the endothermic amount of an endothermic peak during the temperature rise is 0 J / g or 65% or less of the corresponding endothermic amount of a reference liquid consisting of water. (DSC measurement conditions) (1) After holding at 20°C for 1 minute, the temperature was decreased to -80°C at a rate of 5°C / min. (2) After holding at -80°C for 1 minute, the temperature was increased to 20°C at a rate of 10°C / min.

13. The solidified product according to claim 12, which is a solid frozen product.

14. 14. The solidified body according to claim 12 or 13, wherein the intrinsic viscosity (η) of an aqueous polymer solution before solidification, which comprises an aqueous solvent and a water-soluble polymer or a salt thereof, is 0.20 dL / g or more and 0.95 dL / g or less.

15. The solidified body according to any one of claims 12 to 14, comprising the water-soluble polymer in an amount of 0.1 to 20 w / v % or less.

16. The solidified body according to any one of claims 12 to 15, wherein the biological sample is a cell, a tissue, or a tissue-like substance which is a membrane or an aggregate.

17. 17. The solidified body according to claim 12, wherein the biological sample has an orthogonal projection area that is reduced to 1 / 3.5 or more of that before solidification.

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