JP7391528B2 - Polymer aqueous solution for cryopreservation - Google Patents

Description

The present invention relates to an aqueous polymer solution for cryopreservation of biological samples. The present invention also relates to a method of preventing or suppressing the growth of ice crystals in a biological sample using an aqueous polymer solution, and a method of freezing and preserving a biological sample.

With the rapid development of regenerative medicine research in recent years, regenerative medicine such as cell therapy is being actively carried out not only for humans but also in the veterinary field. Bone marrow-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells collected from living bodies are expanded in large quantities after collection and used for regenerative medicine and regenerative medicine research as described above. At this time, it is common to cryopreserve the extra cells and use them as appropriate. Additionally, demand for a stable supply of such cells is increasing.

In the cell cryopreservation mechanism, when ice crystals grow inside cells during the freezing and/or thawing process, cell membranes and intracellular structures are damaged, cell proteins are denatured, and cells are fatally damaged. It is known that this can result in Therefore, when cryopreserving cells, it is important to prevent intracellular freezing. Normally, low-molecular-weight compounds such as methyl sulfoxide (DMSO), glycerin, and propylene glycol are used to permeate into the cells. It is used as a cryoprotective reagent by adding it to a buffer solution such as a culture medium (Patent Document 1). Among these, DMSO is most commonly used, and has a good effect of protecting cells and organelles. However, in intracellular-penetrating cryopreservation solutions, low-molecular-weight compounds that are cryoprotective reagents permeate into cells, so there are concerns about the effects of cryoprotective reagents on cells (Non-Patent Document 1).

Therefore, attempts have been made to use natural cryoprotectants as cryoprotectants instead of chemicals. For example, it is known that disaccharides, oligosaccharides, or polymeric polysaccharides can be added to buffers such as culture media as non-permeating cryoprotective reagents.

Additionally, methods of retaining biological components within the crosslinked body that forms hydrogels are also being considered. Patent Document 2 describes a biological product made from modified hyaluronic acid as a raw material, in which a side chain having a substituent that reacts with a hydroxyl group to form a crosslinked structure is introduced into hyaluronic acid as a raw material with a weight average molecular weight of 50 to 4 million. Preservatives for ingredients are listed. Modified hyaluronic acid reacts with the hydroxyl groups of a compound with multiple hydroxyl groups, such as polyvinyl alcohol, to form a crosslinked product of modified hyaluronic acid, and by embedding biological components in this agar-like hydrogel, it becomes a preservative. is used as. The actual molecular weight of the hydrogel described in Patent Document 2 as a preservative is estimated to be several million or more. In Patent Document 2, biological components are stored in refrigeration at about 4° C., and the storage period is about several days.

Further, Patent Document 3 discloses a cryopreservation composition containing a carboxylated polyamino acid in which the amino groups of the polyamino acid are blocked by being carboxylated (or acetylated) with a carboxylic acid anhydride and an organic amphoteric agent. Are listed. Further, Patent Document 4 discloses fructan as an active ingredient of a cell preservation solution.

Japanese Unexamined Patent Publication No. 63-216476 International Publication No. 2016/076317 Special table 2018-533377 publication JP2012-235728A

REJUVENATION RESEARCH Volume 18 Number 5,2015 Mary Ann Liebert, Inc.

Intracellular cryoprotectants slow down the rate of ice crystal formation within cells and inhibit ice crystal formation by promoting cell dehydration. In particular, DMSO easily penetrates into cells and is therefore effective for cryopreservation of cells with complex structures such as mammalian cells. Chemical substances are cytotoxic. It is thought that as the cryoprotectant penetrates into cells and its intracellular concentration increases, the toxic effect also increases.

Furthermore, 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 has also been reported to affect the differentiation of ES cells (Cryobiology. 2006 Oct;53(2):194-205.). Therefore, it is considered that the use of DMSO as a cryoprotective reagent is not suitable for cell preservation when maintenance of undifferentiated state and functionality of stem cells is required. In addition, when storing samples for a long time using DMSO, it is essential to store the samples in liquid nitrogen or in an atmosphere that requires handling management, which is considered to be an issue for the dissemination of regenerative medicine and regenerative medicine research. It will be done.

On the other hand, natural cryoprotectants such as sugars have an affinity for cells, but their molecular size is large and it is difficult for them to be taken up into cells. Therefore, it is considered that intracellular freezing cannot be sufficiently suppressed just by adding from outside the cells.

As mentioned above, the preservative for biological components described in Patent Document 2 was tested for a storage period of only a few days, and better cryopreservation techniques are required to preserve it for several months. It is believed that there is. In addition, in order to produce the hydrogel described in Patent Document 2, it is necessary to introduce a modifying group into hyaluronic acid, and furthermore, it is necessary to introduce a modifying group into hyaluronic acid, and furthermore, in order to recover biological components from the hydrogel after storage, it is necessary to dissolve the gel. The use of additives is also required. However, although such additives can be used for research tests when introduced into cryopreservation methods, they are difficult to use for actual medical purposes due to the risk of contamination with impurities.

Patent Document 3 discloses a cryopreservation composition containing a carboxylated polyamino acid whose amino group is modified with a carboxylic acid, an organic amphoteric agent, and optionally a polysaccharide. The survival rate was not sufficient.

Furthermore, with the fructan described in Patent Document 4, cell death due to rupture or the like was observed, and cells could not be sufficiently frozen and preserved.

A method of using a non-permeating cryoprotectant reagent is also known, but such a preservation reagent alone does not have a sufficient preservation effect, although the effect on cells is low. Therefore, products are commercially available in which the amount of the compound is reduced or replaced by a combination of an intracellular-permeable compound such as DMSO, glycerin, or propylene glycol, and a non-permeable substance. However, a practical cryoprotectant that exhibits a good cryopreservation effect using only natural ingredients such as biological components has not yet been reported.

The present invention has been made in view of the above problems, and provides an aqueous polymer solution for cryopreservation for appropriately preserving biological samples. In particular, biological samples can be prepared without using chemical substances such as dimethyl sulfoxide (DMSO), propylene glycol (PG), and ethylene glycol (EG), and without adding serum or serum-derived proteins. An aqueous polymer solution for cryopreservation that enables stable long-term storage at temperatures below -27°C, a method for preventing or suppressing the growth of ice crystals in biological samples using the same, and a method for stably preserving biological samples. The purpose is to provide a method for cryopreservation.

The present invention provides an aqueous polymer solution containing a water-soluble polymer or a salt thereof in an aqueous solvent and used for cryopreservation of biological samples, wherein the aqueous polymer solution has an intrinsic viscosity (η) of 0.20 dL/ g or more and 0.95 dL/g or less, and the endothermic peak measured by DSC in the DSC curve of the temperature rising process obtained under the measurement conditions (1) and (2) below using a differential scanning calorimeter (DSC). The present invention relates to an aqueous polymer solution characterized in that no endothermic peak is observed or the temperature at which an endothermic peak is observed is higher than -1.4°C and 1.1°C or lower. Here, the DSC scanning was performed by (1) holding at 20°C for 1 minute, then lowering the temperature to -80°C at a cooling rate of 5°C/min, (2) holding at -80°C for 1 minute, then lowering the temperature to -80°C at 10°C/min. The temperature is raised to 20° C. at a heating rate of min.

Furthermore, in the aqueous polymer solution for cryopreservation of biological samples of the present invention, whether or not an endothermic peak measured by DSC is confirmed in the DSC curve of the heating process obtained under the measurement conditions (1) and (2) above, Or, the temperature at which an endothermic peak is confirmed is above -1.4°C and below 1.1°C, and the orthogonal projected area of the biological sample in the state where the aqueous polymer solution is solidified at -80°C is the same as that before cooling. It is desirable that the area is smaller than the orthogonal projected area of the biological sample in the culture solution (medium).

In addition, the orthogonal projected area of the biological sample is smaller than 1/1 and 1/3.5 or more of the orthographic projected area of the biological sample in the culture solution (medium) before cooling. is desirable.

Moreover, it is preferable that the limiting viscosity (η) of the polymer aqueous solution is 0.32 dL/g or more and 0.65 dL/g or less.

Further, when an endothermic peak is confirmed, it is preferable that the temperature at which the endothermic peak is confirmed is -1.1°C or higher and 0.83°C or lower.

In this specification, "an endothermic peak is not observed" means that "an endothermic peak is not observed" unless an 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 within this range.

As the salt of the water-soluble polymer, for example, a metal salt, a halogen salt, or a sulfate is preferable. As the metal salt, an alkali metal or alkaline earth metal salt is preferable. Sodium, potassium, calcium, etc. are selected as the alkali metal or alkaline earth metal. As the halogen, chlorine, bromine, etc. can be used.

The aqueous polymer solution for cryopreservation of biological samples of the present invention desirably does not contain dimethyl sulfoxide, which is an intracellular-penetrating cryoprotectant. Further, it is preferable that the aqueous polymer solution for cryopreservation of biological samples of the present invention does not contain a cytotoxic cryoprotectant such as ethylene glycol. This is because these are harmful to cells after thawing.

In the aqueous polymer solution of the present invention, in the DSC curve of the temperature decreasing process obtained by the above DSC measurement conditions (1) to (2), the calorific value of the exothermic peak in the temperature decreasing process is 0 J/g, or It is preferable that the calorific value is 65% or less of the corresponding calorific value of the reference solution consisting of.

In the aqueous polymer solution of the present invention, in the DSC curve of the temperature decreasing process obtained by the above DSC measurement conditions (1) to (2), the calorific value of the exothermic peak in the temperature decreasing process is 0 J/g, or It is preferable that the calorific value is 40% or less of the corresponding calorific value of the reference solution consisting of.

Preferably, the aqueous polymer solution contains a water-soluble polymer or a salt thereof in a content of 0.1 w/v% or more and 20 w/v% or less for cryopreservation of biological samples.

Preferably, the water-soluble polymer is an aqueous polymer solution for cryopreservation of biological samples, which is a water-soluble polymer containing sugar residues.

An aqueous polymer solution for cryopreservation of a biological sample in which the biological sample is a cell, a tissue, or a tissue-like substance such as a membrane or an aggregate is preferable.

An aqueous polymer solution for cryopreservation of a biological sample in which the biological sample is a mesenchymal stem cell, a blood cell, an endothelial cell, or a tissue for transplantation is preferable.

Preferably, the biological sample is a polymer aqueous solution for cryopreservation of a biological sample such as a sperm, an egg, or a fertilized egg.

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

The present invention also provides a method for preventing or suppressing the growth of ice crystals in a biological sample during cryopreservation by bringing the biological sample into contact with the aqueous polymer solution for cryopreservation of the biological sample of the present invention. Regarding.

The present invention also provides an aqueous polymer solution containing a water-soluble polymer or a salt thereof in an aqueous solvent and used for cryopreservation of biological samples, wherein the aqueous polymer solution has an intrinsic viscosity (η) of 0. It is 20 dL/g or more and 0.95 dL/g or less, and is measured by differential scanning calorimeter (DSC) in the DSC curve of the heating process obtained under the measurement conditions (1) to (2) below using DSC. A step of including the biological sample in an aqueous polymer solution characterized in that no endothermic peak is observed or the temperature at which an endothermic peak is observed is above -1.4°C and below 1.1°C; The present invention relates to a method for cryopreservation of a biological sample, including a step of cooling and solidifying the aqueous polymer solution containing the biological sample. Here, the DSC scanning was performed by (1) holding at 20°C for 1 minute, then lowering the temperature to -80°C at a cooling rate of 5°C/min, (2) holding at -80°C for 1 minute, then lowering the temperature to -80°C at 10°C/min. The temperature is raised to 20° C. at a heating rate of min.

In addition, in the polymer aqueous solution in the method for cryopreservation of biological samples of the present invention, whether an endothermic peak measured by DSC is confirmed in the DSC curve of the heating process obtained under the measurement conditions (1) and (2) above, or not. Or, the temperature at which an endothermic peak is confirmed is above -1.4°C and below 1.1°C, and the orthogonal projected area of the biological sample in the state where the aqueous polymer solution is solidified at -80°C is the same as that before cooling. It is desirable that the area is smaller than the orthogonal projected area of the biological sample in the culture solution (medium).

In addition, the orthogonal projected area of the biological sample is smaller than 1/1 and 1/3.5 or more of the orthographic projected area of the biological sample in the culture solution (medium) before cooling. is desirable.

Moreover, it is preferable that the limiting viscosity (η) of the polymer aqueous solution is 0.32 dL/g or more and 0.65 dL/g or less.

Further, when an endothermic peak is confirmed, it is preferable that the temperature at which the endothermic peak is confirmed is -1.1°C or higher and 0.83°C or lower.

In this specification, "an endothermic peak is not observed" means that "an endothermic peak is not observed" unless an 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 within this range.

As the salt of the water-soluble polymer, for example, a metal salt, a halogen salt, or a sulfate is preferable. As the metal salt, an alkali metal or alkaline earth metal salt is preferable. Sodium, potassium, calcium, etc. are selected as the alkali metal or alkaline earth metal. As the halogen, chlorine, bromine, etc. can be used.

Further, it is preferable that the aqueous polymer solution for cryopreservation of biological samples of the present invention does not contain cell-permeable and cytotoxic compounds such as dimethyl sulfoxide and ethylene glycol. This is because these are toxic to cells after thawing.

Preferably, the method for cryopreservation of a biological sample further includes a step of preserving the aqueous polymer solution containing the biological sample at a temperature of −27° C. or lower.

The cryopreservation temperature range in the biological sample cryopreservation method of the present invention is not limited as long as it is -27°C or lower, but the upper limit is preferably -70°C or lower, preferably -80°C or lower. It is. The lower limit is desirably -196°C or higher, preferably -150°C or higher.

A method for cryopreservation of a biological sample is preferable, in which the step of cooling and solidifying the aqueous polymer solution containing the biological sample is performed 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 performed by a slow freezing method at a cooling rate of 1° C./min or less.

In the DSC curve of the temperature decreasing process obtained by the above DSC measurement conditions (1) and (2), the calorific value of the exothermic peak of the polymer aqueous solution in the temperature decreasing process is 0 J/g, or the reference solution made of water Preferred is a method for cryopreservation of biological samples that produces less than 65% of the corresponding calorific value.

In the DSC curve of the temperature decreasing process obtained by the above DSC measurement conditions (1) and (2), the calorific value of the exothermic peak of the polymer aqueous solution in the temperature decreasing process is 0 J/g, or the reference solution made of water Preferred is a method for cryopreservation of biological samples that produces 40% or less of the corresponding calorific value.

A method for cryopreservation of a biological sample is preferred, in which the biological sample is a cell, tissue, or tissue-like substance such as a membrane or an aggregate.

A method for cryopreservation of a biological sample in which the biological sample is a sperm, an egg, or a fertilized egg is preferable.

According to the present invention, biological samples such as cells and tissues can be appropriately preserved without using cell-permeable and toxic compounds such as intracellular-permeable cryoprotectants such as DMSO and ethylene glycol. can. Furthermore, since cryopreservation using the water-soluble polymer of the present invention does not permeate into cells, it has low toxicity to cells despite showing a high cryopreservation effect.

Furthermore, since it does not contain serum or serum-derived proteins, it will not be contaminated by bacteria or viruses. Note that it is possible to add proteins that are not contaminated with bacteria or viruses. It is also possible to use cell-permeable and toxic compounds at low concentrations that do not impair cell function.

In addition, the "intrinsic viscosity (η)" of the aqueous solution containing the water-soluble polymer or salt used in the present invention is determined by the following method and calculation formula.

ここで、η_sp：水溶性またはその塩の還元粘度［ｍＬ／ｇ］、η_r：水溶性高分子またはその塩の相対粘度［－］、Ｃ：水溶性高分子またはその塩の濃度［ｇ／ｍＬ］である。
（６）水溶性高分子またはその塩の濃度と、水溶性高分子またはその塩の還元粘度との関係をそれぞれプロットし、近似直線を引く。近似直線の切片（高分子または糖類濃度＝０）の値を水溶性高分子またはその塩の極限粘度とする。 (1) A predetermined amount of NaCl is dissolved in ion-exchanged water at 30°C to prepare a 0.2M NaCl solution (standard solution).
(2) Dissolve a sample of a water-soluble polymer or its salt in a standard solution at 30°C to prepare a stock solution. When a sample of a water-soluble polymer or its salt is obtained in the form of a solution, the solid content 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 sample as is. Even if it is a mixture of water-soluble polymers mixed with impurities, it can be used as a sample. At this time, the viscosity of each of the standard solution and stock solution is measured, and the relative viscosity of the stock solution with respect to the standard solution is adjusted to be 2.0 to 2.4.
(3) Dilute the 30°C stock solution to 5/4, 5/3, and 5/2 times using the 30°C standard solution.
(4) Measure the viscosity of the standard solution, stock solution, and diluted solution at 30°C. An E-type viscometer is used for viscosity measurement.
(5) The relative viscosity (η _r ) is obtained by dividing the viscosity of each of the stock solution and diluted solution by the viscosity of the standard solution, and the reduced viscosity is derived based on the following formula.

Here, η _sp : Reduced viscosity of the water-soluble polymer 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 straight line. The value of the intercept of the approximate straight line (polymer or saccharide concentration = 0) is taken as the limiting viscosity of the water-soluble polymer or its salt.

The aqueous solvent used to dissolve the water-soluble polymer or its salt of the present invention may be water, physiological solution, or sodium ion, potassium ion, An isotonic aqueous solution in which the salt concentration, sugar concentration, etc. are adjusted with calcium ions, etc. is preferable. Specifically, for example, water, physiological saline, phosphate buffered saline (PBS), which is a physiological saline with a buffering effect, Dulbecco's phosphate buffered saline, Tris buffered saline ( Tris Buffered Saline (TBS), HEPES buffered saline, etc., balanced salt solutions such as Hank's balanced salt solution, Ringer's solution, Ringer's lactate, Ringer's acetate, Ringer's bicarbonate, or D-MEM, E-MEM, αMEM, RPMI- Examples include basal media for animal cell culture such as 1640 medium, Ham's F-12, Ham's F-10, and M-199, general culture media for various cells or tissues, and commercially available media. can.

According to the aqueous polymer solution of the present invention, biological samples such as cells can be cryopreserved while maintaining the viability and properties of the biological samples without using cryoprotectants such as highly toxic DMSO or ethylene glycol. can do. The water-soluble polymer of the present invention or its salt, which is a cryoprotectant for the aqueous polymer solution of the present invention and is contained as a solute in the aqueous polymer solution, is not permeable to cell membranes, so it cannot be used when vitrifying biological samples such as cells. It does not penetrate into the sample and swell the living tissue, but rather promotes water removal from cells. In the aqueous polymer solution of the present invention, by adjusting the molecular species and degree of polymerization (molecular weight) of the polymer, and/or by adding a low molecular weight or monomolecular compound to the aqueous solution of the water-soluble polymer, The intrinsic viscosity (η) of the molecular aqueous solution and the temperature and heat amount of the endothermic peak of melting during the heating process are adjusted. In the present invention, the intrinsic viscosity (η) of the polymer aqueous solution is in the range of 0.20 dL/g or more and 0.95 dL/g or less, and no endothermic peak is observed by DSC, or standard water (ultrapure water) is used. By adjusting the temperature so that the endothermic peak appears near the endothermic peak of , the cryopreservation effect of living tissues such as cells is improved.

The aqueous polymer solution for cryopreservation of biological samples of the present invention is a cryopreservation solution that does not penetrate into cells, and does not contain chemical substances that penetrate into cells, such as DMSO or ethylene glycol. Therefore, damage to biological samples, cytotoxicity, and effects on cell properties such as those reported for DMSO can be suppressed. That is, the properties of the biological sample can be maintained during and after cryopreservation.

Furthermore, the aqueous polymer solution for cryopreservation of biological samples of the present invention makes it possible to cryopreserve biological samples while maintaining their viability and properties without using serum and/or serum-derived proteins. Therefore, biological samples are not contaminated with bacteria or viruses.

Furthermore, according to the method for cryopreservation of biological samples and the method for preserving biological samples of the present invention, frozen biological samples can be stored without using cytotoxic chemicals such as DMSO or ethylene glycol, or proteins such as serum. , it can be stored stably for a long period of time at a temperature of -27°C or lower, ie, in a deep freezer. Note that it is possible to add proteins that are not contaminated with bacteria or viruses. It is also possible to use cell-permeable and toxic compounds at low concentrations that do not impair cell function.

The range of 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.

FIG. 3 is a diagram showing the cell survival rate of primary human mesenchymal stem cells during cryopreservation using the test sample solution of the present invention. It is a figure which shows the analysis result of the test sample solution by differential scanning calorimetry. It is an enlarged view showing the analysis results of a test sample solution by differential scanning calorimetry. It is a figure which shows the analysis result of the test sample solution containing ultrapure water by differential scanning calorimetry. It is a figure which shows the analysis result of the test sample solution containing DMSO as a cryoprotectant by differential scanning calorimetry. 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. FIG. 2 is a diagram showing the effect of long-term preservation of primary human mesenchymal stem cells in cryopreservation using the test sample solution of the present invention. FIG. 2 is a diagram showing the effect of long-term preservation of primary human mesenchymal stem cells in refrigerated storage using the test sample solution of the present invention. FIG. 2 is a diagram showing the amount of HGF produced in primary human mesenchymal stem cells after cryopreservation using the test sample solution of the present invention. FIG. 2 is a diagram showing the amount of IL-10 produced in primary human mesenchymal stem cells after cryopreservation using the test sample solution of the present invention. FIG. 2 is a diagram showing the expression levels of undifferentiated biomarkers in primary human mesenchymal stem cells after cryopreservation using the test sample solution of the present invention. It is a figure showing the intracellular vitrification state after freezing in a control test (comparative example 2). FIG. 3 is a diagram showing the state of intracellular vitrification after freezing using the test sample solution of Comparative Example 1. FIG. 3 is a diagram showing the state of intracellular vitrification using the test sample solution of Example 1. It is a figure showing the brightness difference of the intracellular vitrification state. FIG. 3 is a diagram showing the difference in brightness between the intracellular vitrified state and the brightness difference between the solvent and the inside of the cell, which was quantified according to the Munsell brightness scale (0 to 10). It is a figure showing the cryoprotective effect on various cells in cryopreservation using the test sample solution of the present invention. FIG. 2 is a diagram showing the cell survival rate of primary canine mesenchymal stem cells during cryopreservation using low molecular weight saccharides. It is a figure which shows the HPLC analysis result of the test sample by various manufacturing examples. It is a figure which shows the HPLC analysis result of the test sample by various manufacturing examples. It is a figure which shows the HPLC analysis result of the test sample by various manufacturing examples. It is a figure which shows the HPLC analysis result for calibration curves of saccharides which have a predetermined number of sugars. FIG. 3 is a diagram showing the HPLC analysis results of a test sample having a polymer chain of the present invention.

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 a salt thereof, and is 0.20 dL/g or more and 0.95 dL/g or less. It has an intrinsic viscosity (η) in the range of .

Note that 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 from the following method and calculation formula.

ここで、η_sp：水溶性またはその塩の還元粘度［ｍＬ／ｇ］、η_r：水溶性高分子またはその塩の相対粘度［－］、Ｃ：水溶性高分子またはその塩の濃度［ｇ／ｍＬ］である。
（６）水溶性高分子またはその塩の濃度と、水溶性高分子またはその塩の還元粘度との関係をそれぞれプロットし、近似直線を引く。近似直線の切片（高分子または糖類濃度＝０）の値を水溶性高分子またはその塩を含む水溶液の極限粘度とする。 Intrinsic viscosity measurement:
(1) A predetermined amount of NaCl is dissolved in ion-exchanged water at 30°C to prepare a 0.2M NaCl solution (standard solution).
(2) Dissolve a sample of a water-soluble polymer or its salt in a standard solution at 30°C to prepare a stock solution. When a sample of a water-soluble polymer or its salt is obtained in the form of a solution, the solid content 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 sample as is. Furthermore, even if the water-soluble polymer contains impurities, it can be used as a sample as it is. However, this is not the case when measuring the viscosity average molecular weight described below. In the case of a mixed sample containing a plurality of water-soluble polymers, each water-soluble polymer is separated and fractionated, and then the solvent is removed from each solution to obtain a sample of each water-soluble polymer. If the water-soluble polymer is unknown, the substance is identified by HPLC, LC-MS, LC-IR, etc., and the viscosity average molecular weight is calculated as described below. In addition, if multiple unknown water-soluble polymers are included, separate and fractionate each component, identify the substance of each water-soluble polymer by HPLC, LC-MS, LC-IR, etc., and proceed as described below. Calculate the viscosity average molecular weight. Note that even if the mixture contains impurities in the water-soluble polymer, if the viscosity is not affected (for example, impurities such as metal salts), the mixture is used as a sample of the water-soluble polymer. Furthermore, if the sample contains impurities that affect the calculation of the viscosity average molecular weight, the impurities are removed or the water-soluble polymer is fractionated before measurement. Measure the viscosity of each of the standard solution and stock solution, and adjust the relative viscosity of the stock solution to the standard solution to be 2.0 to 2.4.
(3) Dilute the 30°C stock solution to 5/4, 5/3, and 5/2 times using the 30°C standard solution.
(4) Measure the viscosity of the standard solution, stock solution, and diluted solution at 30°C. An E-type viscometer is used for viscosity measurement.
(5) The relative viscosity (η _r ) is obtained by dividing the viscosity of each of the stock solution and diluted solution by the viscosity of the standard solution, and the reduced viscosity is derived based on the following formula.

Here, η _sp : Reduced viscosity of the water-soluble polymer 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 straight line. The value of the intercept of the approximate straight line (polymer or saccharide concentration = 0) is taken as the limiting viscosity of the aqueous solution containing the water-soluble polymer or its salt.

The aqueous polymer 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 aqueous polymer solution of the present invention in the range of 0.20 dL/g or more and 0.95 dL/g or less, the aqueous polymer solution of the present invention can be formed by polymer chains during the cooling process. The aqueous solvent molecules can be well trapped within the matrix. As a result, the molecular movement of water in the aqueous solvent is restricted during cooling, and the water can be solidified and/or frozen in a vitrified state without crystallizing. Normally, in a freezing method called vitrification, when a solution is frozen, solutes (cryoprotectants to prevent frost damage) are removed from the crystals, resulting in an increase in the salt concentration in the remaining solution, which increases the concentration of salt inside and outside cells. This method dehydrates the inside of the cell and vitrifies the inside of the cell by creating an osmotic pressure difference, and is applied to cells whose survival rate after thawing is particularly low. In such a method, in order to make water more easily vitrified, the concentration of a solute (cryoprotectant) is increased or the cooling rate is increased. However, it has been known that increasing the osmotic pressure difference causes greater damage to cells, and that recrystallization occurs during dissolution, resulting in cell damage. It is also technically difficult.

According to the aqueous polymer solution of the present invention, when used as a cryopreservation solution for biological samples such as cells, the inside of the biological sample is dehydrated due to the action of the polymer chains of the water-soluble polymer dissolved in the aqueous polymer solution. This also vitrifies the biological sample, suppressing or preventing the formation of ice crystals within the biological sample, and further weakening the osmotic shock in the biological sample during freezing, which is a problem with conventional vitrification methods. be able to. Therefore, there is no need to contain cytotoxic chemicals such as dimethyl sulfoxide (DMSO) and ethylene glycol, and the aqueous polymer solution of the present invention does not contain DMSO and/or ethylene glycol.

In the aqueous polymer solution containing the water-soluble polymer of the present invention, when frozen, water molecules in the solvent exist in the network matrix of the polymer, resulting in free water or bound water (a state in which water molecules are entangled with the polymer and trapped). It is thought that it exists in the state of Furthermore, it is thought that water molecules that have been excluded from cells etc. due to the osmotic pressure difference are replaced with macromolecules surrounding the biological sample such as cells. In order to achieve 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, making it impossible to entangle and capture water molecules in the water-soluble polymer network. On the other hand, if the intrinsic viscosity of the aqueous polymer solution is too high, replacement with water molecules near the biological sample will not proceed. Further, there may arise a problem that handling properties deteriorate when a biological sample is included in a polymer solution for cryopreservation. By setting the intrinsic viscosity of the polymer aqueous solution in the range of 0.20 dL/g or more and 0.95 dL/g or less, when the polymer aqueous solution of the present invention is cooled, it is in an amorphous glass state in a frozen state. is stabilized. Therefore, cells are not easily damaged by cooling and freezing, and cells can be stably and efficiently cryopreserved. Cell destruction due to ice crystal formation is also less likely to occur. Therefore, according to the present invention, the survival rate of cells in a biological sample after thawing the frozen biological sample is high. Further, for example, it may be preferable that the intrinsic viscosity (η) of the aqueous polymer solution is approximately 0.32 dL/g or more and 0.65 dL/g or less.

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

For example, as the water-soluble polymer in the present invention, a polymer containing a monomer having a hydrophilic group as a repeating unit can be used. Here, the hydrophilic group is, for example, a hydroxyl group, a carboxylic acid group, and a salt thereof. Further, the water-soluble polymer may contain a nitrogen-containing monomer having an optionally substituted amino group or an optionally substituted amide group as a repeating unit. In particular, it may be preferable that the water-soluble polymer has a hydroxyl group at an equatorial position within its structure. This is because it is thought that this allows the solvent water to be better trapped within the matrix formed by the polymer chains during freezing.

A 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 monosaccharides or monosaccharides in which the hydroxyl group and/or hydroxymethyl group of the monosaccharide is substituted, for example, the hydroxyl group and/or hydroxymethyl group may be substituted with a carboxyl group, an amino group, or an N-acetyl group. Examples include, but are not limited to, monosaccharides substituted with at least one substituent selected from the group consisting of an amino group, a sulfoxy group, a methoxycarbonyl group, and a carboxymethyl group.

Examples of monosaccharides include triose, tetrose, pentose, hexose, and heptose. For example, pentoses include ribose, arabinose, xylose, lyxose, xylulose, ribulose, deoxyribose, and the like. Examples of the hexose include glucose, mannose, galactose, fructose, sorbose, tagatose, fucose, fuucrose, rhamnose, and the like.

For example, examples of the monosaccharide substituted with a carboxyl group include uronic acid. Examples of uronic acids include glucuronic acid, iduronic acid, mannuronic acid, and galacturonic acid. Examples of monosaccharides substituted with amino groups include amino sugars. Examples of amino sugars 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 monosaccharides substituted with sulfoxy groups include galactose-3-sulfate. Examples of monosaccharides with multiple substituents include N-acetylglucosamine-4-sulfate, iduronic acid-2-sulfate, glucuronic acid-2-sulfate, N-acetylgalactosamine-4-sulfate, neuraminic acid, and N-acetylglucosamine-4-sulfate. - Examples include acetylneuraminic acid.

For example, the water-soluble polymer in the present invention is a polymer containing monosaccharides as described above as repeating units. For example, the water-soluble polymer in the present invention can be a polymer containing an optionally substituted pentose, hexose, or uronic acid, or a combination thereof as a repeating unit. For example, the water-soluble polymer in the present invention may include a six-membered ring structure in its main chain. This is because polysaccharides and the like having a six-membered ring structure in their main chain may easily form a polymer network structure around cells through hydrogen bonds. Moreover, the water-soluble polymer in 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 in the present invention may be a glycosaminoglycan. It may also be a sulfated polysaccharide in which one or more hydroxyl groups are substituted with a sulfoxy group. Examples of the polymer in the present invention include, but are not limited to, hyaluronic acid, dextran, pullulan, chondroitin sulfate, and the like.

The water-soluble polymer or its salt in the present invention may be of natural origin, or may be chemically synthesized. Commercially available water-soluble polymers or their salts may be used as they are. Using a naturally derived polymer compound or a commercially available polymer compound with a larger molecular weight, the cleavage product is obtained by subjecting it to treatments such as hydrolysis, enzyme treatment, and subcritical treatment, and the molecular weight is adjusted. It may also be used as a water-soluble polymer in the invention. Furthermore, each monomer may be naturally derived, a naturally derived monomer may be modified or substituted, or it may be chemically synthesized. For example, preferably, the monomer contained in the water-soluble polymer in the present invention is a biological component. It is thought that an aqueous polymer solution containing a water-soluble polymer has low cytotoxicity when used as a cryopreservation solution for biological samples. In addition, processes required for conventional cryopreservation solutions, such as washing and removing the cryoprotectant contained in the cryopreservation solution from the thawed biological sample, may be unnecessary or simplified.

The water-soluble polymer or its salt in the present invention has, for example, a viscosity average molecular weight of more than 3,000 and less than 500,000. By using a water-soluble polymer or a salt thereof having a viscosity average molecular weight of this level, when the aqueous solution is cooled, the amorphous glass state of the solvent in the frozen state is stabilized. Therefore, cells are not easily damaged by cooling and freezing, and cells can be stably and efficiently cryopreserved. Therefore, the survival rate of cells in the biological sample is high after the biological sample is thawed after cryopreservation. If the viscosity average molecular weight of the water-soluble polymer is 3000 or less, vitrification may be difficult to occur satisfactorily. Furthermore, if the viscosity average molecular weight of the water-soluble polymer is greater than 500,000, the viscosity will increase significantly, and the solubility may decrease, and the prepared solution may foam, resulting in poor handling properties. For example, the viscosity average molecular weight is preferably 10,000 or more. Further, a polymer having a viscosity average molecular weight of 400,000 or less, more preferably 200,000 or less is preferable, and 150,000 or less is particularly preferable. This is because the viscosity can be adjusted to be low as an aqueous solution and it is easy to handle.

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 from the above method and formula. required by

上記のマークホーイング桜田の式に、測定で導出した極限粘度と、文献等で公開されているＫとαの値から粘度平均分子量Ｍを求める。例えば、水溶性高分子がヒアルロン酸の場合、Ｋ＝３．６×１０^-4およびα＝０．７８である。その他、プルランおよびゼラチンの場合、文献値よりＫ＝９×１０^-4、α＝０．５であり、デキストランでは、Ｋ＝６．３×１０^-8、α＝１．４、また、コンドロイチン硫酸では、Ｋ＝５．８×１０^-4、α＝０．７４、フルクタンでは、Ｋ＝１．６×１０^-3、α＝０．４５、カルボキシル化ポリリジンはＫ＝２．７８×１０^-5、α＝０．８７である。 Viscosity average molecular weight:
The viscosity average molecular weight is calculated from the intrinsic viscosity.

The viscosity average molecular weight M is determined using the Mark Hoing Sakurada equation described above, the intrinsic viscosity derived through measurement, and the values of K and α published in literature. For example, when the water-soluble polymer is hyaluronic acid, K=3.6×10 ⁻⁴ and α=0.78. In addition, in the case of pullulan and gelatin, K = 9 × 10 ^-4 and α = 0.5 from literature values, and for dextran, K = 6.3 × 10 ^-8 , α = 1.4, and chondroitin sulfate. Then, K = 5.8 x 10 ^-4 , α = 0.74, for fructan, K = 1.6 x 10 ^-3 , α = 0.45, and for carboxylated polylysine, K = 2.78 x 10 ^-5 , α=0.87.

K and α are numerical values that vary depending on the type of polymer. For example, the values of K and α are disclosed in many published documents such as "Polymer Materials Handbook" (edited by the Society of Polymer Science and Technology), and have not been published. The viscosity average molecular weight can be calculated from the intrinsic viscosity (η) using the value shown in FIG.

In the present invention, the molecular weight calculated by this method is defined as the viscosity average molecular weight. In addition, in the case of monosaccharides, disaccharides, and compounds considered to be single molecules, such as sucrose and glucuronic acid, the molecular weight can be clearly specified from the structural formula, so the molecular weight specified from the structural formula can be assumed to be the viscosity average molecular weight. Treat it as such.

The hydrophilic groups of the water-soluble polymer or its salt in the present invention are unmodified or, even if modified, are less than 50% of the total number of hydrophilic groups, that is, no substituents have been introduced into the polymer chain. Or, even if introduced, it is desirable that the amount is 50% or less of the total number of hydrophilic groups. It is assumed that the hydrophilic groups of water-soluble polymers, especially OH groups, _NH2 groups, and COOH groups, contribute to the protection of biological samples, vitrification of solvents, and replacement with water molecules around biological samples. This is because it is advantageous to improve the survival rate of biological samples if these functional groups are not modified. Furthermore, when attempting to introduce functional groups into water-soluble polymers, the reagents used to modify the hydrophilic groups of the polymers may adversely affect the cryoprotective effect of the polymers or There is a risk that it may become unusable as a cryopreservation solution for cryopreservation of samples.

Examples of the water-soluble polymer salts used in the present invention include metal salts, halogen salts, and sulfates. The metal salt is preferably an alkali metal or alkaline earth metal salt. Sodium, potassium, calcium, etc. are selected as the alkali metal or alkaline earth metal. As the halogen, chlorine, bromine, etc. can be used.

The aqueous solvent for the aqueous polymer solution of the present invention may be water, physiological solution, or sodium ion, potassium ion, calcium ion, etc. to adjust the salt concentration and sugar content so that the osmotic pressure is almost the same as that of body fluids or cell fluids. An isotonic aqueous solution with adjusted concentration etc. is preferred. Specifically, for example, water, physiological saline, phosphate buffered saline (PBS), which is a physiological saline with a buffering effect, Dulbecco's phosphate buffered saline, Tris buffered saline ( Examples include, but are not limited to, Tris Buffered Saline (TBS), HEPES buffered saline, balanced salt solutions such as Hank's balanced salt solution, Ringer's solution, lactated Ringer's solution, acetic Ringer's solution, bicarbonate Ringer's solution, and the like. Further, the solvent may contain other optional components such as an isotonic agent, a chelating agent, and a solubilizing agent, as long as the effects of the present invention are not impaired. As used herein, "optional component" means a component that may or may not be included. For example, the solvent may be a 5% aqueous glucose solution. Furthermore, a cell culture medium 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 includes, for example, commercially available media, D-MEM, E-MEM, αMEM, RPMI-1640 medium, Ham's F-12, Ham's F-10, M-199, etc. Examples include basal media for animal cell culture, and general culture media for various cells or tissues. Therefore, when a biological sample is cryopreserved using the aqueous polymer solution of the present invention, the biological sample such as cultured cells may be suspended in the aqueous polymer solution of the present invention and cryopreserved; The cryopreservation step may be performed by adding the water-soluble polymer of the invention or a salt thereof at a desired concentration to a culture solution containing a biological sample, such as a cultured cell culture solution or a cell suspension.

Further, the pH of the aqueous polymer solution of the present invention may be adjusted as necessary. For example, when the hydrophilic group of a monomer having a hydrophilic group is a carboxylic acid group, an aqueous polymer solution containing a water-soluble polymer obtained by polymerizing such a monomer may exhibit acidity. In such a case, by adjusting the pH to make the aqueous polymer solution a neutral solution, the solution can become more suitable for the survival of cells to be cryopreserved, and it is thought that the survival rate of the cells will improve. The salt used for pH adjustment is not limited, and salts commonly used for adjusting the pH of aqueous solutions can be used.

The aqueous polymer solution for cryopreservation of biological samples of the present invention has the following characteristics: The measured endothermic peak is not confirmed, or the temperature at which the endothermic peak is confirmed exceeds -1.4°C and is 1.1°C or less.
DSC measurement conditions:
(1) After holding at 20°C for 1 minute, lower the temperature to -80°C at a cooling rate of 5°C/min, and (2) After holding at -80°C for 1 minute, lower the temperature to 20°C at a heating rate of 10°C/min. Temperature rising.

That is, in the polymer aqueous solution of the present invention, the endothermic peak estimated to be caused by the melting of water is not observed during the temperature raising process of the polymer aqueous solution, or if it is observed, the endothermic peak is estimated to be caused by the melting of water, or if it is observed, the endothermic peak is estimated to be caused by the melting of water. 1.1° C. or lower, that is, near the temperature (0.7° C.) at which an endothermic peak is observed in ultrapure water under DSC measurement under the same conditions. For vitrification within biological samples such as solvents and cells, it generally appears desirable to use a cryopreservation solution with a lower melting point (freezing point). However, the present inventors have found that in order to achieve good vitrification of biological samples during freezing, it is desirable for the cryopreservation solution to have characteristics opposite to this common knowledge. Normally, the phenomenon that the melting point (freezing point) is lower than that of water occurs when solutes are dispersed in the matrix of water, but in the aqueous polymer solution of the present invention, on the contrary, Water molecules exist in a polymer network matrix in an aqueous solvent formed of polymers, and either water exists as free water without freezing in the matrix, or even if water freezes, it is a polymer network matrix. Freezes when entangled in water (bound water). Therefore, in the aqueous polymer solution of the present invention, it is presumed that the endothermic peak of water melting is not observed during the temperature raising process, or that it is observed at a temperature close to the temperature at which the endothermic peak of pure water is observed.

Furthermore, in the present invention, the present inventors have discovered that in order to adjust the endothermic peak, a saccharide having a molecular weight lower than that of the water-soluble polymer or a salt thereof is added to an aqueous polymer solution containing the water-soluble polymer of the present invention or a salt thereof. It has been found that this can be achieved by adding

The low molecular weight saccharide or its salt is held within the polymer matrix by hydrogen bonding by the hydrophilic groups in the water-soluble polymer in the present invention. It is thought that when polymers holding low molecular weight sugars or their salts are present around biological samples such as cells, water molecules near the cell membrane are replaced with low molecular weight sugars. Therefore, it is thought that water discharged from a biological sample such as cells during freezing due to the osmotic pressure difference replaces not only the macromolecules surrounding the biological sample such as cells but also low molecular weight sugars. Therefore, the addition of low molecular weight sugars or their salts is believed to aid in the drainage of water from within the biological sample and, through replacement with water molecules, suppress or prevent the formation and growth of ice crystals near cell membranes. By setting the intrinsic viscosity of the aqueous polymer solution of the present invention in the range of 0.20 dL/g or more and 0.95 dL/g or less, replacement of water molecules and low molecular weight saccharides can be promoted. As a result, cell membrane damage during freezing can be significantly suppressed. In other words, a low molecular weight saccharide or a salt thereof can function as a component for cell protection in the aqueous polymer solution of the present invention. If the intrinsic viscosity of the aqueous polymer solution of the present invention is too small, low molecular weight saccharides will diffuse into the polymer solution, making it impossible to efficiently entangle and trap water molecules in the water-soluble polymer network. On the other hand, if the intrinsic viscosity of the aqueous polymer solution is too high, it is thought that the replacement of low molecular weight sugars with water molecules will not proceed efficiently.

Since the drainage of water from within the biological sample is promoted and cells are protected, biological samples such as cells frozen in the aqueous polymer solution of the present invention are more effective than biological samples before freezing. Its volume contracts. For example, the orthogonal projected area of a biological sample solidified at -80°C in the aqueous polymer solution of the present invention is compared to the orthogonal projected area of a biological sample in a culture solution (medium) before cooling (before freezing). , it has been reduced to about 1/3.5 or more and less than 1/1. When a biological sample is frozen using the aqueous polymer solution of the present invention, the inside of the cell is well dehydrated during freezing. As a result, damage to cells during freezing is significantly reduced, and thawed cells exhibit a high survival rate.

In the present invention, the "orthogonal projected area" of a biological sample refers to the area of the biological sample in the image taken from above in the normal direction of the cooling stage when the biological sample is observed on a cooling stage for a microscope. It means the area calculated using the image analysis software ImageJ (https://imagej.nih.gov/ij/).

In order to retain low molecular weight saccharides or salts thereof within the matrix, the water-soluble polymer of the present invention or its salt hydrophilic groups may be unmodified, or even if modified, the total number of hydrophilic groups may be reduced. In other words, it is desirable that no substituents be introduced into the polymer chain, or even if substituents are introduced, it should be 50% or less of the total number of hydrophilic groups. If the hydrophilic groups of water-soluble polymers are modified, the retention effect of low molecular weight sugars or their salts by the hydrophilic groups will be reduced, and even if low molecular weight sugars coexist, biological samples will not survive. In some cases, it may not be possible to make a sufficient contribution to improving the rate. Therefore, for example, it may not be preferable to modify the OH group or NH ₂ group of a water-soluble polymer with a carboxylic acid or the like.

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, a disaccharide, or an oligosaccharide having a viscosity average molecular weight of 1000 or less.

The low molecular weight saccharide in the present invention may be, for example, the monosaccharide mentioned above as a monomer constituting the water-soluble polymer in the present invention. For example, the low molecular weight saccharide is glucose, fructose, galactose, or an amino sugar in which the alcohol group is oxidized with uronic acid or the alcohol group is substituted with an amino group, sucrose, trehalose, or a polymer or combination thereof. . Furthermore, the low molecular weight saccharide may be, for example, a fragment of a polymer in the present invention, such as hyaluronic acid, dextran, pullulan, or chondroitin sulfate. Although not particularly limited as long as it does not impair the effects of the present invention, low molecular weight saccharides include, for example, cleavage products (fragments) of glycosaminoglycans, that is, monosaccharides constituting glycosaminoglycans, disaccharides thereof, or It can be an oligosaccharide. In the case of hyaluronic acid, it is desirable to use one with a viscosity average molecular weight of 1000 or less.

Preferably, the low molecular weight saccharide is a cleavage product of hyaluronic acid. Therefore, preferably, the low molecular weight saccharide or salt thereof in the present invention is glucuronic acid or N-acetylglucosamine, or a disaccharide or oligo consisting thereof, or a salt thereof. Preferably, the saccharide may be glucuronic acid or a modified compound thereof, or a disaccharide or oligosaccharide thereof, or a salt thereof.

In the present invention, the term "cleavage product" refers to a compound with a molecular weight smaller than the original polymer, which is thought to be obtained when a polymer is subjected to treatments such as hydrolysis, enzyme treatment, or subcritical treatment. means. For example, the polymer in the present invention may be a water-soluble polymer obtained by processing a larger polymer compound, as described above, and the low molecular weight saccharide in the present invention may be a water-soluble polymer obtained by processing a larger polymer compound. It may also be a low molecular weight saccharide obtained by the treatment of In the present invention, the cleavage products can be monomers that are constituents of the original polymer and/or polymers of different degrees of polymerization of the monomers and/or mixtures thereof.

"Subcritical treatment" means bringing into contact a subcritical fluid as an extraction solvent brought into a subcritical state under conditions of a predetermined temperature and a predetermined pressure, and a raw material to be extracted. For example, when water is raised to a pressure of 22.12 MPa or higher and a temperature of 374.15° C. or higher, it becomes neither liquid nor gas. This point is called the critical point of water, and hot water with a temperature and pressure lower than the critical point is called subcritical water. By using the hydrolysis action of this subcritical water, desired components can be obtained from the raw material to be extracted. In the present invention, the conditions for subcritical treatment are, for example, a temperature of 150°C or higher and 350°C or lower, and the subcritical treatment pressure can be higher than or equal to the saturated vapor pressure at each temperature, but for example, 0. It can be set to .5 MPa or more and 25 MPa or less. After the subcritical treatment, components having a predetermined molecular weight or less can be separated and recovered and used as the cleavage product in the present invention. Furthermore, the hydrolysis and enzymatic treatments are not particularly limited, and commonly used reagents and treatment methods can be used without any problem.

The water-soluble polymer and low molecular weight saccharide in the present invention may be obtained simultaneously through a single subcritical treatment. That is, the water-soluble polymer and low molecular weight saccharide in the present invention have a first molecular weight distribution in a molecular weight range of more than 3000 and less than 500000 as a viscosity average molecular weight, and a molecular weight range of 3000 or less as a viscosity average molecular weight. It may also be a subcritically processed product of a polymer compound having a second molecular weight distribution. Therefore, for example, in the method for producing an aqueous polymer solution of the present invention, a polymer which is a polysaccharide having a viscosity average molecular weight of more than 500,000 is dissolved in water, and then extracted under subcritical conditions of the water. The method may also include a step of obtaining the water-soluble polymer and low molecular weight saccharide of the present invention. However, of course, the water-soluble polymer and low molecular weight saccharide in the present invention may be treated and/or separated on the basis of molecular weight in separate processing steps and used in combination.

In the aqueous solution of a low molecular weight saccharide or a salt thereof in the present invention, an endothermic peak is observed at a temperature lower than the temperature (0.7° C.) at which an endothermic peak is observed in DSC measurement in pure water. For example, hyaluronic acid with a viscosity average molecular weight of 1000 or less has an endothermic peak temperature of -8.1°C, which is lower than the endothermic peak temperature of pure water, 0.7°C.

On the other hand, the water-soluble polymer or its salt in the present invention is, for example, hyaluronic acid with a viscosity average molecular weight of 15,000 and an endothermic peak temperature of -1.4°C, and pullulan with a viscosity average molecular weight of 373,000 at 1.49°C. be. According to the present invention, when such a water-soluble polymer or its salt is combined with a low molecular weight saccharide or its salt, the endothermic peak temperature of the aqueous polymer solution is brought close to that of pure water, or the endothermic peak temperature is itself is no longer observed in DSC measurements. For example, when hyaluronic acid with a viscosity average molecular weight of 1,000 or less is added to hyaluronic acid with a viscosity average molecular weight of 15,000, 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 1,000 or less is added to pullulan with a viscosity average molecular weight of 373,000, the endothermic peak temperature decreases to 0.83°C.

In other words, if hyaluronic acid with a viscosity average molecular weight of 1000 or less is mixed with a water-soluble polymer whose endothermic peak temperature is lower than that of pure water, the endothermic peak temperature will rise and approach the endothermic peak temperature of pure water. , or can be adjusted so that the endothermic peak itself does not appear in DSC. In addition, when hyaluronic acid with a viscosity average molecular weight of 1000 or less is mixed with a water-soluble polymer whose endothermic peak temperature is higher than that of pure water, the endothermic peak temperature decreases and can approach the endothermic peak temperature of pure water. Or, it is adjusted so that the endothermic peak itself does not appear in DSC. In this way, for example, by using hyaluronic acid having a viscosity average molecular weight of 1000 or less, the endothermic peak temperature of the water-soluble polymer can be adjusted to be around 0.7° C., the endothermic peak temperature of pure water.

The low molecular weight saccharide or salt thereof of the present invention that exhibits such effects is not limited to the above-mentioned hyaluronic acid having a viscosity average molecular weight of 1000 or less. As long as the viscosity average molecular weight is 1000 or less, the same effect can be achieved not only with hyaluronic acid but also with other saccharides such as sucrose and trehalose. The low molecular weight saccharide in the present invention either exists between water molecules and inhibits the freezing of water, or works with a network of water-soluble polymers to trap water within the network (bound water or free water). The polymer aqueous solution of the present invention may be acting to realize behavior close to that of pure water by inhibiting the ice crystallization of water in the first place. Guessed.

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 has a DSC temperature-lowering process obtained under the following measurement conditions (1) and (2). In the curve, the calorific value of the exothermic peak in the temperature-lowering process (calorific value during temperature-lowering) is 0 J/g or 65% or less of the corresponding calorific value of the reference solution consisting of water, preferably consisting of water. It is 40% or less of the corresponding calorific value of the reference solution.
DSC measurement conditions:
(1) After holding at 20°C for 1 minute, lower the temperature to -80°C at a cooling rate of 5°C/min, and (2) After holding at -80°C for 1 minute, lower the temperature to 20°C at a heating rate of 10°C/min. Temperature rising.

The fact that the calorific value of the exothermic peak during the temperature-lowering process in the DSC measurement is smaller (less than 100%) than the corresponding calorific value of the reference solution consisting of water in the polymer aqueous solution of the present invention means that the frozen polymer aqueous solution This means that there remains bound water and free water that do not form ice crystals. That is, in the aqueous polymer solution of the present invention, replacement of water-soluble polymers and/or low-molecular-weight saccharides with water progresses, and water molecules are trapped within the matrix of water-soluble polymers, thereby inhibiting the molecular movement of water. It is restricted. This promotes the dehydration of water from within the cells and allows the water to solidify in a glassy state without crystallizing. Good vitrification within the aqueous polymer solution and the biological sample contained therein is achieved. Therefore, in cryopreservation of biological samples using the aqueous polymer solution of the present invention, cell destruction due to the formation of ice crystals can be suppressed or prevented. Furthermore, in the aqueous polymer solution of the present invention, not only the formation of ice crystals is suppressed or prevented during the cooling process, but also recrystallization of water during the subsequent heating process, that is, during melting can be suppressed. That is, it is considered that the aqueous polymer solution of the present invention stabilizes the glass state of the biological sample in the frozen state. Since the glassy state is more stabilized in this way, the aqueous polymer solution of the present invention can provide high cell protection without the need for the addition of human or bovine serum or protein components such as serum-derived components (e.g. albumin). Show effectiveness. Therefore, there is no need to worry about infectious diseases, and it is thought that it will not be affected by lot-to-lot differences due to biological preparations. Note that it is possible to add proteins that are not contaminated with bacteria or viruses.

Since the aqueous polymer solution of the present invention does not contain a cell-permeable compound, it is considered to have low cytotoxicity when it comes into contact with a biological sample for cryopreservation of the biological sample. Furthermore, since there is no need to increase the concentration of solutes to limit the movement of water molecules, damage to cells is thought to be low. In addition, the low molecular weight saccharide or its salt of the present invention that is added has a cell protective effect that rapidly replaces the water excreted from inside the cell around the cell during freezing, so it does not affect the properties of the cell during cryopreservation. It is thought that it will not change. Therefore, it is considered that the aqueous polymer solution of the present invention allows biological samples to be cryopreserved while maintaining cell characteristics.

Furthermore, unlike conventional vitrification methods, the aqueous polymer solution of the present invention does not require a large cooling rate to reduce osmotic shock during cooling. Therefore, according to the aqueous polymer solution of the present invention, biological samples can be efficiently cryopreserved by a simple method with reduced toxicity, and thawed cells exhibit a high survival rate after cryopreservation.

The aqueous polymer solution of the present invention contains a water-soluble polymer or its salt 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 satisfactorily. For example, the concentration of the water-soluble polymer or its salt is preferably 1 w/v% or more, particularly preferably 5 w/v% or more. Furthermore, if the concentration is higher than 20 w/v%, the viscosity may become too high and the handling properties may deteriorate. Preferably, the concentration of the polymer or its salt is 5 w/v% or more and 20 w/v% or less.

The aqueous polymer solution of the present invention contains a low molecular weight saccharide or a salt 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. Preferably. If the concentration of the saccharide or its salt is too low, the cell protection effect of the low molecular weight saccharide may not be sufficiently obtained. Further, even if saccharides are added at a concentration of 1/10 times or more that of the water-soluble polymer, it is difficult to obtain further effects as a cell-protecting component.

According to the results of thermal analysis of the aqueous polymer solution of the present invention by differential scanning calorimetry (DSC), the aqueous polymer solution of the present invention has a glass transition temperature around -23°C±4°C. As described above, in the aqueous polymer solution of the present invention, vitrification is achieved by trapping water molecules in a network of water-soluble polymers during freezing. Therefore, high cooling rates are not required for cell protection during cooling as required by conventional vitrification methods. For this reason, for example, the aqueous polymer solution of the present invention containing a biological sample is cooled to −27° C. or lower, which is the glass transition point temperature of the aqueous polymer solution of the present invention, after the biological sample is included in the aqueous polymer solution of the present invention. Cells, tissues, etc. to be preserved can be stably frozen and preserved by simply placing them in a freezing treatment container or the like and leaving them in a -80°C deep freezer. Since a low temperature of -150° C., which is required for normal cryopreservation of cells, is not necessarily required, it is not necessary to prepare a special container or liquid nitrogen for liquid nitrogen cryopreservation. Therefore, operations related to cryopreservation of biological samples can be significantly simplified. In addition, the aqueous polymer solution of the present invention can also suppress recrystallization of water during thawing, so if the aqueous polymer solution of the present invention is used, a series of steps of freezing, preserving, and thawing a biological sample can be performed. , can be performed easily and efficiently without the need for special techniques. Of course, it is also possible to cryopreserve a biological sample in liquid nitrogen or liquid nitrogen vapor using the aqueous polymer solution of the present invention.

Since the aqueous polymer solution of the present invention functions as a non-permeable cryopreservation solution, there are no particular limitations on biological samples that can be cryopreserved using the aqueous polymer solution of the present invention. It can be used for cryopreservation of various types of cells. Furthermore, the species of origin of the cells is not particularly limited. Since the aqueous polymer solution of the present invention can effectively suppress or prevent ice crystal formation and recrystallization during freezing and thawing, it can be favorably used for mammalian cells with complex structures. Therefore, it can be applied to the cryopreservation of cells of various animal species, such as mice, dogs, and humans. Furthermore, it is known that the aqueous polymer solution of the present invention is more susceptible to freezing problems than ordinary cultured cells, and it seems that the so-called conventional slow freezing method will inevitably lead to a significant decrease in survival rate. It can be particularly suitably used for cryopreservation of reproductive cells such as stem cells, early embryos, eggs, sperm, and fertilized eggs. Furthermore, since the aqueous polymer solution of the present invention does not contain chemical substances such as DMSO and ethylene glycol that can act as differentiation factors, it can be used for preserving cells that need to be maintained undifferentiated, for example, for regenerative medicine applications. stem cells can also be cryopreserved without the risk of differentiation.

Furthermore, since no serum or serum-derived proteins are added, there is no contamination by bacteria or viruses. Note that it is possible to add proteins that are not contaminated with bacteria or viruses. It is also possible to use cell-permeable and toxic compounds at low concentrations that do not impair cell function.

In addition, the aqueous polymer solution of the present invention has an excellent effect of stabilizing the glass state in a frozen state, so it can be used for cells such as eggs and fertilized eggs, which are large in size and are known to be difficult to preserve. It can also be used for cryopreservation of organized cell structures, tissues, and tissue-like materials, such as tissues obtained through regenerative medicine.

That is, the aqueous polymer solution of the present invention can be used for biological samples selected from cells, tissues, tissue-like materials such as membranes or aggregates, and can achieve a high survival rate. In particular, the aqueous polymer solution of the present invention can be used regardless of primary cells or established cells; mesenchymal stem cells; hematopoietic stem cells; neural stem cells; somatic stem cells such as bone marrow stem cells and germ stem cells; blood cells; endothelial cells; In particular, it can be advantageously used in the cryopreservation of primate stem cells, which are said to have lower freezing tolerance than mice, the cryopreservation of tissues for transplantation, and the cryopreservation of germ cells in reproductive medicine.

Furthermore, if a water-soluble polymer that is a biological component is used as the water-soluble polymer of the present invention, for example, it is possible to thaw a frozen biological sample and use it as it is as a solution for cell administration. . Furthermore, in the case of tissue or tissue-like material, it is also possible to add such an aqueous polymer solution of the present invention at the stage of organization and freeze-preserve it as it is. This is thought to reduce post-transplant problems. In such cases, the preferred water-soluble polymer for the aqueous polymer solution of the present invention is hyaluronic acid. In particular, hyaluronic acid has a viscosity average molecular weight of greater than 3,000, more preferably greater than 5,000, and less than 60,000, more preferably less than 20,000.

The cryopreservation method of the present invention includes a step of including a biological sample in the aqueous polymer solution used for cryopreservation of the biological sample of the present invention, and a step of cooling and solidifying the aqueous polymer solution containing the biological sample. Contains.

The aqueous polymer solution of the present invention used in the cryopreservation method 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 the DSC curve of the heating process obtained using a differential scanning calorimeter (DSC) under the measurement conditions (1) and (2) below, the endothermic peak measured by DSC is not confirmed, or the endothermic peak is The temperature at which the
(1) After holding at 20°C for 1 minute, lower the temperature to -80°C at a cooling rate of 5°C/min, and (2) After holding at -80°C for 1 minute, lower the temperature to 20°C at a heating rate of 10°C/min. Temperature rising. ). The orthogonal projected area of a biological sample solidified at -80°C in an aqueous polymer solution is 1/1 compared to the orthogonal projected area of a biological sample in a culture solution (medium) before cooling (before freezing). It has been reduced to 3.5 or more and less than 1/1.

The aqueous polymer solution of the present invention used in the cryopreservation method of the present invention has a glass transition temperature around -23°C ± 4°C; The cryopreservation method of the present invention does not require a large cooling rate because water molecules are entangled and trapped in the network. For this reason, for example, the aqueous polymer solution of the present invention containing a biological sample is cooled to −27° C. or lower, which is the glass transition point temperature of the aqueous polymer solution of the present invention, after the biological sample is included in the aqueous polymer solution of the present invention. Cells and tissues to be preserved can be stably frozen and preserved while maintaining their viability by simply placing them in a freezing treatment container and leaving them in a deep freezer at -80°C. .

However, the cryopreservation temperature range 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.

The cryopreservation method 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 thought that at this cooling rate, the inside of the cell is appropriately dehydrated, the intracellular fluid is vitrified well, and a high cell cryoprotective effect is obtained.

Furthermore, the pH of the aqueous polymer solution of the present invention may be adjusted as necessary before it is brought into contact with a biological sample. For example, when the hydrophilic group of a monomer having a hydrophilic group is a carboxylic acid group, an aqueous polymer solution containing a water-soluble polymer obtained by polymerizing such a monomer may exhibit acidity. Further, when the saccharide has a carboxylic acid group or the like, the aqueous polymer solution can become acidic due to such saccharide. In such a case, by adjusting the pH to make the aqueous polymer solution a neutral solution, the solution can become more suitable for the survival of cells to be cryopreserved, and it is thought that the survival rate of the cells will improve. The salt used for pH adjustment is not limited, and salts commonly used for adjusting the pH of aqueous solutions can be used.

In the cryopreservation method for biological samples using the aqueous polymer solution of the present invention, the vitrification state is stabilized and toxicity is low, so biological samples can be stored stably for a long period of time. In this specification, long-term stable storage means, for example, that when the aqueous polymer solution of the present invention is used, the survival rate of a biological sample, such as a cell, after thawing is based on the cell viability immediately before storage. A decrease of less than 10%, preferably less than 5% after 5 months, or a decrease of less than 20%, preferably less than 10% after 6 months, or a decrease of less than 15% after 12 months. This means that a reduction of only about 30%, preferably less than 30%, is observed. In addition, in this specification, long-term stable storage means, for example, when cells are frozen, stored at -80°C for a long period of time, thawed, and then stored at 4°C, This means that even after 24 hours, there was a decrease in viability of less than 5% based on the cell viability immediately after thawing. It is believed that the cryopreservation method using the aqueous polymer solution of the present invention allows cells to be cryopreserved under less stressful conditions than the conventional cryopreservation method that uses a DMSO solution as a cryopreservation solution. Therefore, in the cryopreservation method of the present invention, a significantly higher cell survival rate after thawing can be obtained compared to a cryopreservation method using a conventional cryopreservation solution such as a 10% DMSO solution. Furthermore, not only cells immediately after thawing, but also cells that are refrigerated after thawing can exhibit a high cell survival rate. According to the cryopreservation method using an aqueous polymer solution of the present invention, cells can be stably cryopreserved for a long period of time without changing the properties of the cells.

The present invention will be specifically explained based on Examples, but the present invention is not limited thereto. Further, in the following, hyaluronic acid manufactured by Lifecore Biomedical is sodium hyaluronate, but for the sake of simplicity, it is referred to as "hyaluronic acid".

Water-soluble polymers and aqueous polymer solutions for cryopreservation <Preparation of test samples and sample solutions>
・Example 1
Polymer hyaluronic acid with an average molecular weight of 1 million (manufactured by Shanghai Easier Industrial Development Co., Ltd.) and water were mixed at a ratio of 20:100 in a pressure-resistant container with a volume of 2 L, and the treatment temperature was 175°C, the treatment pressure was 0.89 MPa, and the treatment time was 3. Subcritical treatment was performed in minutes. Thereafter, the subcritically treated product was dried by freeze-drying (this drying could also be done by spray drying). As a result, the water-soluble polymer of the present invention, which is a mixture of high molecular weight hyaluronic acid having a viscosity average molecular weight of about 10,000 and hyaluronic acid having a viscosity average molecular weight of 1000, was obtained.

When this water-soluble polymer was confirmed by HPLC (Figure 12), the test sample of Example 1 was a mixture of high molecular weight hyaluronic acid decomposition products and low molecular weight hyaluronic acid decomposition products obtained by subcritical treatment of hyaluronic acid. Therefore, high molecular weight hyaluronic acid was precipitated in ethanol, low molecular weight hyaluronic acid was fractionated into the supernatant, and this fractionated supernatant was reanalyzed by HPLC. The viscosity average molecular weight of the low molecular weight hyaluronic acid component obtained in Example 1 was estimated to be 1000 because it roughly matched the HPLC peaks of hyaluronic acid in Production Examples 1 to 3 (FIGS. 11A to 11C), which will be described later. .

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

A test sample solution of Example 1 was obtained by dissolving 1 g of the test sample of Example 1 in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent is water) (i.e. , the concentration of the hyaluronic acid sample in the test sample solution was 10 w/v%). Note that in Examples, Production Examples, and Comparative Examples of the present invention, including this Example 1, αMEM medium is used as a solvent, but ultrapure water may be used as a solvent instead of αMEM medium. Furthermore, in the DSC measurement described below, ultrapure water was used instead of αMEM as a solvent. Note that each test sample obtained in the following examples was prepared and used at a predetermined concentration using an appropriate solvent in each evaluation test described below.

・Manufacturing example 1
A test sample of hyaluronic acid fragments having a viscosity average molecular weight of 1000 was obtained by performing the same operation as in Example 1, except that the subcritical treatment was performed for a treatment time of 7 minutes. The intrinsic viscosity was 0.08 dL/g.

・Manufacturing example 2
A test sample of hyaluronic acid fragments having a viscosity average molecular weight of 2000 was obtained by performing the same operation as in Example 1, except that the subcritical treatment was performed for a treatment time of 5 minutes. The intrinsic viscosity was 0.14 dL/g.

・Manufacturing example 3
A test sample of hyaluronic acid fragments having a viscosity average molecular weight of 3000 was obtained by performing the same operation as in Example 1, except that the subcritical treatment was performed for a treatment time of 4 minutes. The intrinsic viscosity was 0.19 dL/g.

・Comparative example 1
DMSO (manufactured by Nacalai Tesque Co., Ltd., cell culture grade) was used as a test sample. A test sample solution of Comparative Example 1 was obtained by dissolving 1 mL of this test sample in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, solvent is water) (i.e., test sample The DMSO concentration of the test sample in the solution was 10 w/v%).

・Comparative example 2
αMEM medium (manufactured by Gibco, product number C1257-1500BT, solvent is water) used as a solvent was used as a test sample.

・Comparative example 3
The test sample of Comparative Example 3 was prepared by dissolving 1 g of gelatin (viscosity average molecular weight 315,000, manufactured by Nitta Gelatin Co., Ltd.) in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent is water) as a solvent. A solution was obtained (ie, the gelatin concentration of the test sample in the test sample solution was 10% w/v). The intrinsic viscosity was 0.50 dL/g.

・Comparative example 4
Comparative Example 4 was obtained by dissolving 1 g of high molecular weight hyaluronic acid (manufactured by Shanghai Easier Industrial Development Co., Ltd.) with a viscosity average molecular weight of 1,000,000 in 100 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent is water) as a solvent. A test sample solution was obtained (that is, the hyaluronic acid concentration of the test sample in the test sample solution was 1 w/v%). The intrinsic viscosity was 17.2 dL/g.

・Comparative example 5
Hyaluronic acid (manufactured by Lifecore Biomedical; powder) having a viscosity average molecular weight of 15,000 was used as a test sample. A test sample solution of Comparative Example 5 was obtained by dissolving 1 g of this test sample in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, solvent is water) (i.e., test sample The hyaluronic acid concentration of the test sample in the solution was 10 w/v%). The intrinsic viscosity was 0.65 dL/g.

・Comparative example 6
Chondroitin sulfate sodium salt A (manufactured by Sigma) having a viscosity average molecular weight of 23,000 was used as a test sample. A test sample solution of Comparative Example 6 was obtained by dissolving 1 g of this test sample in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, solvent is water) (i.e., test sample The chondroitin sulfate concentration of the test sample in the solution was 10 w/v%). The intrinsic viscosity was 0.97 dL/g

・Comparative example 7
The test sample of Production Example 1 (hyaluronic acid fragment sample with a viscosity average molecular weight of 1000) was added to carboxypolylysine in which 60% of amino groups were carboxylated (viscosity average molecular weight 13400: manufactured by Bio Verde Co., Ltd., CryoScarless DMSO free). It was added in an amount of 1 w/v% to obtain a test sample solution of Comparative Example 7. The intrinsic viscosity was 0.11 dL/g.

・Comparative example 8
Fructan derived from rakkyo was used as a test sample. Note that fructan was obtained by extraction and purification in the following manner according to Example 2 of JP-A No. 2012-235728.

Approximately 1 kg of dried rakkyo as a raw material was crushed, mixed with extraction water in which 15 g of calcium hydroxide was suspended, extracted at room temperature for 1 hour, then heated at 95°C for 1 hour, Thereafter, the mixture was extracted by heating at 60° C. overnight. The extract was deodorized by adding activated carbon, and then filtered through diatomaceous earth to remove the residue and activated carbon. The obtained extract was passed through an ultrafiltration membrane to purify fructans.

The obtained purified liquid was once heated and sterilized, and then activated carbon was added to perform the decolorization treatment again. This purified liquid was sterilized by heating at 85° C. for 1 hour, freeze-dried, and powdered to obtain 400 g of purified fructan powder.

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

A test sample solution of Comparative Example 8 was obtained by dissolving 1 g of this fructan powder in 10 mL of αMEM medium as a solvent (that is, the fructan concentration of the test sample in the test sample solution was 10 w/v%). The intrinsic viscosity was 0.11 dL/g

・Comparative example 9
Pullulan (manufactured by Tokyo Chemical Industry Co., Ltd.) having a viscosity average molecular weight of 373,000 was used as a test sample. A test sample solution of Comparative Example 9 was obtained by dissolving 1 g of this test sample in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, solvent is water) (i.e., test sample solution The hyaluronic acid concentration of the test sample inside is 10 w/v%). The intrinsic viscosity was 0.55 dL/g.

・Comparative example 10
Hyaluronic acid concentration 10 w/v% in which 1 g of the test sample of Comparative Example 5 (hyaluronic acid with a viscosity average molecular weight of 15000) was dissolved in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, solvent is water). A test sample solution of Comparative Example 10 was obtained by adding the test sample of Production Example 2 (a hyaluronic acid fragment sample with a viscosity average molecular weight of 2000) at a final concentration of 1 w/v% to the aqueous solution of Comparative Example 10. The intrinsic viscosity was 0.47 dL/g.

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

・Example 3
A test at a pullulan concentration of 10 w/v% in which 1 g of 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 (manufactured by Gibco, product number C1257-1500BT, the solvent is water). The test sample of Production Example 1 (hyaluronic acid fragment sample with a viscosity average molecular weight of 1000) was added to the test sample solution of Example 3 at a final concentration of 1 w/v%. The intrinsic viscosity was 0.55 dL/g.

・Example 4
A test at a pullulan concentration of 10 w/v% in which 1 g of 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 (manufactured by Gibco, product number C1257-1500BT, the solvent is water). The test sample of Production Example 1 (hyaluronic acid fragment sample with a viscosity average molecular weight of 1000) was added to the test sample solution at a final concentration of 5 w/v%. The pH of the obtained aqueous solution was adjusted to neutral using 10mM Tris-HCl to obtain a test sample solution of Example 4. The intrinsic viscosity was 0.55 dL/g.

・Example 5
Polymer hyaluronic acid with an average molecular weight of 1 million (manufactured by Shanghai Easier Industrial Development Co., Ltd.) and water were mixed at a ratio of 20:100 in a pressure-resistant container with a volume of 2 L, and the treatment temperature was 175°C, the treatment pressure was 0.89 MPa, and the treatment time was 3. Subcritical treatment was performed in .5 minutes. Thereafter, the subcritically treated product was dried by freeze drying or spray drying. As a result, a hyaluronic acid sample having a viscosity average molecular weight of about 6,000 was obtained.

Sucrose (Nacalai Tesque Co., Ltd.) is added to a test sample solution with a hyaluronic acid concentration of 10 w/v% in which 1 g of this hyaluronic acid is dissolved in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent is water). ) and glucuronic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., molecular weight (viscosity average molecular weight and fictitious) 194) were added at a final concentration of 1 w/v%. A cryopreservation solution for testing in Example 5 was obtained. The intrinsic viscosity was 0.32 dL/g.

<Test Example 1: Cryopreservation using test sample solution of primary human mesenchymal stem cells and evaluation of its preservation effect>
Cultured primary human mesenchymal stem cells (Lonza PT2501) were added to the test sample solutions of Examples 1 and 5 and Comparative Examples 1, 2, 4, 5, 7, and 8 at a concentration of 1 × 10 ⁶ cells/mL ( (serum-free).

Thereafter, the cell suspension containing each test sample solution was frozen in a -80°C freezer in a slow cell freezer (Nalgene® Mr. Frosty). Cell suspensions containing frozen test samples were stored at -80°C for 7 days and then quickly thawed in a 37°C bath. Immediately after thawing, the cell suspension containing each test sample solution was evaluated for cell viability by trypan blue staining. The results are shown in FIG. 1 and Table 1 (Table 1 only shows the results of Example 5).

Figure 1 shows cryopreservation in a medium to which no cryopreservation solution has been added, that is, 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. The cell survival rate after thawing is shown. As shown in FIG. 1, in Example 1, that is, the hyaluronic acid obtained by subcritical treatment has a viscosity average molecular weight of about 10,000, and further has a viscosity average molecular weight of 1000, that is, smaller than 10,000. In Example 1, the hyaluronic acid sample containing a cleavage product of hyaluronic acid having a molecular weight is a water-soluble polymer in an aqueous polymer solution, the intrinsic viscosity is 0.49 dL/g, and no endothermic peak is confirmed. A significantly high preservation effect of over 90% was obtained with the test sample solution. The preservation effect of this test sample solution was even higher than that of Comparative Example 1, which contained DMSO, which is a commonly used cryopreservative, as a cryopreservative. It can be seen that the aqueous polymer solution of the present invention is an excellent cryopreservation solution with low cytotoxicity. The test sample solution of Comparative Example 4 contains high molecular weight hyaluronic acid with a large molecular weight of 1,000,000 as a test sample, but has a large intrinsic viscosity of 17.2 dL/g, and although no endothermic peak is observed, it does not protect cells. Almost no effect was observed. Note that in Comparative Example 2, which did not contain a cryopreservative, the cell survival rate was approximately 0%. In addition, in Comparative Example 7, which contains carboxypolylysine as a test sample and has a small intrinsic viscosity of 0.11 dL/g, the water molecules that moved outside the cells and the macromolecules or low molecular weight sugars around the cells diffused. It is thought that the replacement of water molecules with polymers or low molecular weight sugars does not proceed. In addition, since the endothermic temperature peak appears at a temperature lower than -1.4°C, it is thought that the freezing rate is slow and fine ice crystals are generated, causing cells to swell, and as a result, cell tissue was destroyed, so the cell protection effect was not as good as in Example 1 or Comparative Example 1 in which DMSO was used as a cryoprotectant.

Furthermore, Comparative Example 8 using fructan also had a small intrinsic viscosity of 0.11 dL/g, and the replacement of water molecules that moved outside the cells with macromolecules or low-molecular-weight sugars, etc. around the cells did not proceed. Furthermore, since the endothermic temperature peak appears on the lower temperature side than -1.4°C, the freezing rate is slow and fine ice crystals are generated, resulting in about half of the cells bursting and dying.

It is understood 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 about 6000 is -1. 4°C).

In addition, the aqueous polymer 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. After suspending the cells in the culture medium, they can be frozen and stored. It is considered that there is no need to centrifuge the cells to be stored, and it is possible to maintain higher cell survival rates and cell properties.

<Test Example 2: Analysis of each test sample solution by differential scanning calorimeter>
The solvent of each test sample solution of Examples 1 to 5, Comparative Examples 1 to 10, and Production Examples 1 to 3 was changed from αMEM medium to ultrapure water to prepare samples for differential scanning calorimetry analysis. Each sample was scanned with a differential scanning calorimeter (DSC) as described below.
(1) After holding at 20℃ for 1 minute, lower the temperature to -80℃ without speed.
(2) After holding at -80°C for 1 minute, raise the temperature to 20°C at a temperature increase rate of 10°C/min.

FIG. 2A shows DSC curves of the heating process obtained under the above DSC measurement conditions for Example 1, Comparative Example 5, and ultrapure water (Comparative Example 2). FIG. 2B is an enlarged view of FIG. 2A near the glass transition temperature of the aqueous polymer solution of the present invention. Further, the DSC curves of Example 1, Comparative Example 1, and ultrapure water (Comparative Example 2) are shown in FIGS. 3A to 3C.

In addition, the temperature at which an endothermic peak estimated to be caused by the melting of water is observed in the DSC curve during the temperature rising process of each test sample solution, and the calorific value of the exothermic peak observed in the DSC curve during the temperature cooling process. analyzed. The results are shown in Table 1. In addition, in Table 1, the calorific value of the exothermic peak is shown as a ratio (%) when the calorific value of 106 J/g measured with ultrapure water (Comparative Example 2; used as a reference liquid) is taken as 100.

As shown in Figures 2A and 2B, in Comparative Example 2 where the test sample was ultrapure water (i.e., the 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 the temperature of the glass at around -20°C was observed. Metastasis was confirmed (Fig. 2B). On the other hand, in the test sample of Example 1 containing hyaluronic acid with a viscosity average molecular weight of about 10,000 and a cleavage product of hyaluronic acid (low molecular weight hyaluronic acid with a viscosity average molecular weight of 1000), the peak of ice crystal melting was It was extremely small, almost not observed (FIG. 2A), and only a glass transition was observed around -23°C±4°C (FIG. 2B). This result shows that in the aqueous polymer solution of Example 1, water exists in the matrix in a non-frozen state (free water) or in a state entangled with the polymer and trapped (bound water), and This shows a significantly advantageous effect of the aqueous polymer solution of the present invention in that recrystallization of water upon melting is suppressed.

Furthermore, as shown in FIG. 3A, in Comparative Example 2 in which the test sample was ultrapure water, a very large exothermic peak accompanying the formation of ice crystals was observed during the temperature cooling process. Also, during the temperature increase process, a large amount of heat of fusion was observed due to the melting of ice crystals. In Comparative Example 1 in which the test sample was DMSO, as shown in FIG. 3B, only a peak associated with the formation of small ice crystals 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 a 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 no ice crystals were observed. The peak associated with the formation was also much smaller than the peak observed in Comparative Example 1. Therefore, in the polymer aqueous solution containing the test sample of Example 1, almost no ice crystals were formed, and it is considered that it was in an extremely good vitrification state.

In addition, the temperature at which an endothermic peak was observed (endothermic peak temperature) was -1.4°C in Comparative Example 5 (the test sample was hyaluronic acid with a viscosity average molecular weight of 15,000); In Example 2, in which hyaluronic acid fragments with a viscosity average molecular weight of 1000 or less were mixed, the temperature was -0.4°C, which was close to the endothermic peak appearance temperature of pure water, 0.7°C. The endothermic peak temperature of Production Example 1 is -8.1°C, and if it is added to a water-soluble polymer whose endothermic peak appearance temperature is lower than pure water, it is expected that the endothermic peak appearance temperature will further decrease. In addition, the endothermic peak appearance temperature can be increased to approach the endothermic peak appearance temperature of pure water, or it can be adjusted so that the endothermic peak or exothermic peak itself does not appear.

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, which was 0.7°C. When this Comparative Example 9 is mixed with Production Example 1 (endothermic peak appearance temperature is -8.1°C) (Examples 3 and 4), the endothermic peak appearance temperature is lowered rather than increased, and the pure water The endothermic peak appearance temperature was 0.83°C, which is close to 0.7°C.

In other words, when hyaluronic acid with a molecular weight of 1,000 or less is mixed with a water-soluble polymer whose endothermic peak appearance temperature is lower than that of pure water, the peak appearance temperature of the polymer aqueous solution increases and becomes closer to that of pure water, and the hyaluronic acid with a molecular weight of about 1,000 and It has become clear that when a water-soluble polymer whose endothermic peak appearance temperature is higher than that of pure water is mixed, the peak appearance temperature of the aqueous polymer solution is lowered and can be brought closer to that of pure water.

On the other hand, the endothermic peak temperature of the hyaluronic acid fragment 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 appearance temperature of Comparative Example 10, in which the low molecular weight sample of Production Example 2 was mixed with Comparative Example 5 (endothermic peak appearance temperature is -1.4°C), was lowered to -1.6°C.

This result shows that the endothermic peak temperature of the aqueous polymer solution can be adjusted by using a low molecular weight saccharide or a salt thereof, particularly preferably a low molecular weight saccharide with a viscosity average molecular weight of 1000 or less. This shows that it is possible to make the endothermic peak temperature of pure water around 0.7°C. When sucrose, trehalose, or glucuronic acid was used as a low-molecular sugar or its salt having a viscosity average molecular weight of 1000 or less, the same effect on the endothermic peak temperature as in the sample of Production Example 1 was observed.

Further, the calorific value of the exothermic peak during the temperature cooling process of the test sample solutions of Examples 1 to 5 was smaller than the corresponding calorific value of the reference solution made of water (Comparative Example 2), and was 65% or less. It can be seen that in the aqueous polymer solution of the present invention, water is not crystallized but solidified in a glass state.

The endothermic peak temperatures of the test sample solutions of Examples 1 to 5 are around 0.7°C, the endothermic peak temperature of water, that is, they exhibit behavior similar to pure water, and the calorific value of the exothermic peak is that of water. The fact that the calorific value of the exothermic peak observed during the temperature cooling process is smaller than that of the exothermic peak 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 within its matrix upon freezing.

<Test Example 3: Evaluation of long-term preservation effect in cryopreservation using test sample solution of primary human mesenchymal stem cells>
Cultured primary human mesenchymal stem cells (Lonza PT2501) were suspended in the test sample solutions (serum-free) of Example 1 and Comparative Example 1 at a concentration of 1×10 ⁶ cells/mL. Thereafter, the cell suspension containing each test sample solution was frozen in a -80°C freezer in a slow cell freezer (Nalgene® Mr. Frosty). After storing the frozen cell suspension containing each test sample solution at -80°C for up to 12 months, take out the cell suspension containing each test sample solution at the specified storage period and store at 37°C. It was rapidly thawed in a hot bath. Immediately after thawing, the cell viability of the cell suspension containing each test sample solution was evaluated by trypan blue staining. The results are shown in Figure 4A. In addition, cell suspensions that had been 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 suspension after storage at 4°C was evaluated by trypan blue staining. Note that the cell survival rate after storage at 4°C was calculated based on the cell survival rate immediately after thawing (that is, immediately before storage) as 100%. The results are shown in Figure 4B.

From the results shown in Figure 4A, when cryopreserved using the aqueous polymer solution of the present invention, the frozen cells showed a high cell survival rate of over 95%, which remained almost unchanged even after 5 months of storage. You can see that On the other hand, in the cryopreservation using the sample solution containing the test sample (DMSO) of Comparative Example 1, a decrease in cell viability was already observed after two months of cryopreservation. It can be seen that after 3 months, the cell survival rate in the cryopreservation of Comparative Example 1 was below 50%. This result was in contrast to the aqueous polymer solution of the present invention, which still maintained a high cell survival rate even after 3 months. After 6 months of cryopreservation, cryopreservation using the polymer aqueous solution of the present invention resulted in a decrease in survival rate of less than 10%, while cryopreservation using the sample solution of Comparative Example 1 showed that the cell survival rate decreased by less than 10%. The survival rate had dropped to about 25%. Furthermore, in cryopreservation using the aqueous polymer solution of the present invention, a high cell viability was still maintained even after 12 months of cryopreservation, and the decrease in cell viability was only less than 15%.

Therefore, it can be seen that according to the aqueous polymer solution of the present invention, cells can be cryopreserved stably for a long period of time with a high cell survival rate.

Furthermore, when cells stored for a long period of time at -80°C were thawed and then stored at 4°C, when the polymer aqueous solution of the present invention was used, the cell survival rate after one day of storage at 4°C was lower than that just before storage. While the cell survival rate was close to 100% in Comparative Example 1, it was reduced to about 60%. Cells cryopreserved using the aqueous polymer solution of the present invention still had a cell survival rate of 40% or more even after storage at 4°C for one week. This is due to the fact that during cryopreservation using the aqueous polymer solution of the present invention, the cells that survived after thawing were cells that proceeded to proliferate normally, and that the aqueous polymer solution of the present invention was used to preserve the cells during storage. This indicates that the aqueous polymer solution of the present invention has an extremely excellent cell preservation effect.

<Test Example 4: Evaluation of properties after cryopreservation using test sample solution of primary human mesenchymal stem cells>
Cultured primary human mesenchymal stem cells (Lonza PT2501) were suspended in the test sample solutions (serum-free) of Example 1 and Comparative Example 1 at a concentration of 1×10 ⁶ cells/mL. Thereafter, the cell suspension containing each test sample solution was frozen in a -80°C freezer in a slow cell freezer (Nalgene® Mr. Frosty). Cell suspensions containing each frozen test sample solution were stored frozen at -80°C for 6 months. After 6 months, the cell suspension containing each test sample solution was rapidly thawed in a 37°C hot bath, washed with αMEM, and then seeded at ^2.5 x 10 cells into a 6-well plate, and 4 days later. , HGF and IL-10 concentrations in the culture medium were quantified. For the quantification of HGF and IL-10, 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 were used, respectively. (manufactured by S.D., Inc.) according to the procedure in the procedure manual attached to the kit. The results are shown in Figures 5A and 5B.

As shown in FIG. 5A, when cryopreserved in the presence of the sample solution of Comparative Example 1 containing DMSO as a cryopreservative, HGF production in cells after thawing was high. On the other hand, when hyaluronic acid with a viscosity average molecular weight of 10,000 containing a cleavage product of hyaluronic acid was cryopreserved using the test sample solution of Example 1 of the present invention, the amount of HGF produced in cells after thawing was low; It was less than 1/3 of that. The results in the presence of DMSO indicate that the cells were under stress upon storage in the presence of DMSO. Such high HGF production is not observed when cells are preserved using an aqueous polymer solution containing the water-soluble polymer of the present invention, indicating that cells are stably protected in the aqueous polymer solution of the present invention.

As shown in FIG. 5B, the IL-10 production amount was high in the cells cryopreserved using the test sample solution of Example 1, and the IL-10 production amount was high in the cells cryopreserved using the test sample solution of Comparative Example 1. It was very low. This result shows that the aqueous polymer solution of the present invention can favorably freeze-preserve cells while maintaining cell functions.

Since the aqueous polymer solution of the present invention does not contain cytotoxic compounds such as DMSO or ethylene glycol, the aqueous polymer solution of the present invention does not expose biological samples to stress conditions, unlike conventional cryopreservation solutions. It has the remarkable effect of being able to be cryopreserved while maintaining its properties.

Furthermore, since no serum or serum-derived proteins are added, there is no contamination by bacteria or viruses. Note that it is possible to add proteins that are not contaminated with bacteria or viruses. It is also possible to use cell-permeable and toxic compounds at low concentrations that do not impair cell function.

<Test Example 5: Evaluation of properties (undifferentiated property) after cryopreservation using test sample solution of primary human mesenchymal stem cells>
Cultured primary human mesenchymal stem cells (Lonza PT2501) were suspended in the test sample solutions (serum-free) of Example 1 and Comparative Example 1 at a concentration of 1×10 ⁶ cells/mL. Thereafter, the cell suspension containing each test sample solution was frozen in a -80°C freezer in a slow cell freezer (Nalgene® Mr. Frosty). Cell suspensions containing each frozen test sample solution were stored frozen at -80°C for 6 months. After 6 months, the cell suspension containing each test sample solution was taken out and rapidly thawed in a 37°C hot bath. In the cell suspension containing each test sample solution after thawing, the expression intensity of CD90, CD44, and CD105 was analyzed by flow cytometry. The results are shown in FIG.

CD90, CD44, and CD105 are typical surface proteins expressed on undifferentiated mesenchymal stem cells, and are used as undifferentiated markers of mesenchymal stem cells. As shown in Figure 6, the expression of all undifferentiated biomarkers, CD90, CD44, and CD105, was significantly lower than that of cells cryopreserved with the test sample solution of Comparative Example 1 in cells cryopreserved with the aqueous polymer solution of Example 1. It was higher than that of the cells. Therefore, it can be seen that the cells cryopreserved with the aqueous polymer solution of Example 1 maintain the expression of undifferentiated markers. That is, cells cryopreserved with 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 differentiation state that occurs when the aqueous test solution of Comparative Example 1 is used.

These results show that the cryopreservation method using the aqueous polymer solution of the present invention is suitably applicable to the cryopreservation of stem cells, which is important to be cryopreserved in an undifferentiated state.

<Test Example 6: Evaluation of a polymer aqueous solution containing various samples as a cryopreservation solution>
Cultured primary canine mesenchymal stem cells (cyagen C160) were added to the test sample solutions of Examples 1 to 4 and Comparative Examples 3, 6, 9, and 10 (serum-free) at a concentration of 1×10 ⁶ cells/mL. (However, the concentration of the hyaluronic acid sample in the test sample solution of Example 1 was 20 w/v%). Thereafter, the cell suspension containing each test sample solution was frozen in a -80°C freezer in a slow cell freezer (Nalgene® Mr. Frosty). After cryopreservation for one day, the cell suspension containing each test sample solution was taken out and rapidly thawed in a 37°C hot bath. The cell viability of the cell suspension containing each test sample solution after thawing was evaluated by trypan blue staining. The results are shown in Table 1.

From the cell viability results shown in Table 1 together with the results of Test Example 1, the water-soluble polymer of the present invention is not limited to hyaluronic acid, and even other water-soluble polymers can be used. It can be seen that if the molecular aqueous solution has a predetermined intrinsic viscosity and endothermic peak temperature, it exhibits a high cell protection effect. 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 by using a water-soluble polymer in combination with a saccharide or its salt having a smaller molecular weight, It was found that the polymer aqueous solution showed good cell protection effect. As shown in the results of Test Example 2, by adding a low-molecular sugar or its salt to an aqueous polymer solution, the endothermic peak temperature of the sample solution was reduced to 0.7°C, the endothermic peak temperature of pure water. It is presumed that this was due to the adjustment being made so that they were close to each other. Comparative Example 10 is a test sample in which the low molecular weight saccharide of Production Example 2 (hyaluronic acid fragment sample with a viscosity average molecular weight of 2000) was added to a polymer aqueous solution of hyaluronic acid with a viscosity average molecular weight of 15000 (Comparative Example 5). Although it is a solution, as shown in the results of Test Example 2, the endothermic peak temperature of the DSC curve is -1.6 °C, lower than the endothermic peak temperature of Comparative Example 5 (-1.4 °C). As a result, the endothermic peak appearance temperature of pure water has moved away from 0.7°C. Furthermore, in Test Example 6 as well, in Comparative Example 10, a cell survival rate as high as that in Example was not obtained. From this result, it is important to adjust the endothermic peak temperature of the test sample solution to the desired range by mixing low molecular weight saccharides or their salts with the aqueous polymer solution, which will result in a high cell survival rate. I found out that I can get it.

In addition, if the prepared test sample solution is acidic due to the addition of sugars or its salts, a good cell protection effect can be obtained by making the test sample solution neutral by adjusting the pH. Obtained. It is thought that pH adjustment resulted in more appropriate conditions for cell survival. In order for the added low molecular weight saccharide or its salt to act as a cell protection component and further improve the cell viability of cells after cryopreservation, the low molecular weight saccharide or its salt should be added to the polymer aqueous solution. It was necessary that it be added in an amount of 1 to 5 w/v% relative to the water-soluble polymer. On the other hand, even when the amount of low molecular weight saccharide or its salt added was greater than 10 w/v% relative to the water-soluble polymer, no further influence on the effect of improving cell survival rate was observed.

From Table 1, 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 the endothermic peak temperature of the DSC curve is -1.4°C. Hereinafter, it can be seen that when the temperature is higher than 1.1°C, the cell survival rate after cryopreservation decreases. As is clear from Table 1, it can be seen that even if only one of the limiting viscosity and endothermic peak temperature is within a predetermined range, the desired effect (high cell survival rate) cannot be obtained. By having an intrinsic viscosity (η) within the predetermined range mentioned above, the polymer solution helps expel water molecules from within the biological sample, and efficiently transfers the expelled water molecules to the high concentration surrounding the biological sample. It is contemplated that molecules and/or small molecule sugars can be substituted. Furthermore, the fact that the endothermic peak temperature exceeds -1.4°C and is approximately 1.1°C or less is thought to indicate that the polymer solution can retain water molecules within the polymer matrix during freezing. It will be done.

Furthermore, the results of Test Example 6 revealed that the aqueous polymer solution of the present invention exhibits a good cryoprotective effect not only on human mesenchymal stem cells but also on canine mesenchymal stem cells.

<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 suspended in the test sample solutions (serum-free) of Example 1 and Comparative Example 1 at a concentration of 1×10 ⁶ cells/mL. As a control, a control suspension in which cells were suspended in the test sample solution of Comparative Example 2 consisting of αMEM medium containing no cryopreservative was similarly prepared. Thereafter, 5 μL of the cell suspension containing each test sample solution and the control suspension were added to a hard glass sample plate (16φ x 0.12 mm), and a hard glass cover glass (12φ x 0.12 mm) was added. The temperature was lowered to −80° C. at a rate of 5° C./min using a linKam cooling stage for microscopes (THMS600), and images were taken at −80° C. using a transmitted light microscope (Olympus BX53). The results are shown in Figures 7A-C.

FIG. 7A shows the results for the control suspension, and it can be seen that when using only the medium, the cells turned dark due to the formation of ice crystals within the cells and diffuse reflection of light. FIG. 7B shows the results of Comparative Example 1 in which DMSO was the test sample. In FIG. 7B as well, the cells have turned dark and it can be seen that micro ice crystals have been formed. FIG. 7C shows the results of the polymer aqueous solution containing the test sample of Example 1. It can be seen that the cells have turned bright and the inside of the cells has become vitrified to an amorphous state.

This result shows that the aqueous polymer solution of the present invention freezes the inside of cells by vitrifying them. It was also found that by including sugars, which are cleavage products of hyaluronic acid, a glassy state can be formed more stably. It is thought that vitrification occurred stably and efficiently because the formation of ice crystals around the cells was suppressed by sugars having low molecular weights.

<Test Example 8: Evaluation of intracellular vitrification state using brightness difference>
Cultured primary canine mesenchymal stem cells (cyagen C160) were suspended in the test sample solutions (serum-free) of Examples 1 to 5 and Comparative Examples 1 to 10 at a concentration of 1 x ¹⁰ cells/mL. . As a control, a control suspension in which cells were suspended in the test sample solution of Comparative Example 2 consisting of αMEM medium containing no cryopreservative was similarly prepared. Thereafter, 5 μL of the cell suspension containing each test sample solution and the control suspension were added to a hard glass sample plate (16φ x 0.12 mm), and a hard glass cover glass (12φ x 0.12 mm) was added. The temperature was lowered to −80° C. at a rate of 5° C./min using a linKam cooling stage for microscopes (THMS600), and images were taken at −80° C. using a transmitted light microscope (Olympus BX53). The results of Example 1 and Comparative Examples 1 and 2 are shown in FIGS. 8A and 8B.

The difference in brightness (absolute value) between the inside and outside of the cell in the observed image shown in FIG. 8A was analyzed using image analysis software ImageJ. In each image, read the brightness of the solvent region, the brightness inside the cell, and the Munsell brightness under the same conditions, and compare the read data of the brightness of the solvent region and the brightness inside the cell with the read data of each Munsell brightness. Then, the Munsell brightness of the closest reading 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 value). If the read value of the brightness of the intracellular region or solvent region read by the image analysis software is a value darker than the read value of the darkest Munsell brightness value of 0, for convenience, the Munsell value of the intracellular region or solvent region If the value is set to 0, and the read value of the brightness of the intracellular region or solvent region read by the image analysis software is a value brighter than the read value of the brightest value of 10 of the Munsell brightness, for convenience, Let the Munsell lightness value of the region be 10.

As a result of the measurement, in the control suspension (Comparative Example 2), the Munsell brightness in the solvent region was 5, the Munsell brightness in the intracellular region was 0, and the difference in Munsell brightness between the solvent region and the intracellular region was 5. . In Comparative Example 1, the brightness of the solvent region was 4, the Munsell brightness of the intracellular region was 0, and the Munsell brightness difference was 4. In contrast, in Example 1, the Munsell brightness in the solvent region was 4, the Munsell value in the intracellular region was 2, and the Munsell brightness difference was 2 (FIG. 8B). For Examples 2 to 5 and Comparative Examples 3 to 10, the Munsell brightness of the solvent region and the Munsell brightness of the intracellular region were similarly determined, and the difference in brightness was calculated. The results are shown in Table 1.

When ice crystals form during freezing, visible light is blocked and brightness decreases. Therefore, it is preferable that the Munsell lightness is 2 or more in the solvent region and the intracellular region. This level of Munsell brightness indicates that no ice crystals are formed in the solvent region and the intracellular region, that is, the region is well vitrified. In Example 1, the Munsell brightness of the solvent region was 4, the frozen cells had turned bright, and the Munsell value of the intracellular region was 2. Therefore, it was found that no ice crystals were formed in either region.

Further, the difference in brightness between the Munsell value in the solvent region and the Munsell value in the intracellular region was 2 in Examples 2 to 5, as in Example 1. That is, in the example, the brightness of the intracellular region and the brightness of the solvent region are close to each other. This result shows that in cryopreservation using the polymer aqueous solution containing the test sample of Example, the frozen state is almost the same in the matrix region and the intracellular region, that is, ice crystals are present in the region inside the biological sample. It shows that it is not formed. It is desirable that the difference in brightness between the intracellular region and the solvent region is 3 or less. Furthermore, it is desirable that the brightness value of the solvent region is greater than or equal to the brightness value of the intracellular region.

<Test Example 9: Evaluation of cells in frozen sample solution>
Cultured primary canine mesenchymal stem cells (cyagen C160) were added to the test sample solutions of Examples 1 to 5, Comparative Examples 1 to 10, and Production Examples 1 to 3 (serum-free) at a concentration of 1×10 ⁶ cells/mL. (containing). Thereafter, 5 μL of the cell suspension containing each test sample solution was added to a hard glass sample plate (16φ x 0.12 mm), covered with a hard glass cover glass (12φ x 0.12 mm), and placed on a linKam plate. The temperature was lowered to −80° C. at a rate of 5° C./min to solidify using a cooling stage for a microscope (THMS600) manufactured by the company, and the mixture was observed using a transmitted light microscope (Olympus BX53). Images were taken from above in the normal direction of the cooling stage before the temperature was lowered (before solidification) and after the temperature was lowered to -80°C (solidified state). The cell area of each cell was calculated using image analysis software ImageJ in the images taken before and after the temperature was lowered (orthogonal projected area of the cell), and changes in the orthogonal projected area of the cell due to solidification were determined. The ratio of the area of the cells after solidification to the area of the cells before solidification was calculated and taken as the cell contraction rate (%). The results are shown in Table 1.

Cells frozen with the test sample solutions of Examples 1 to 5 showed cell shrinkage rates of 38%, 33%, 33%, 33%, and 40%, respectively. In cryopreservation using the test sample solutions of Examples 1 to 5, drainage of water from the frozen cells is promoted, and the cells are well dehydrated during freezing. In the freezing of biological samples with the aqueous polymer solutions of Examples 1 to 5, the water-soluble polymer and/or low molecular weight saccharide of the present invention is present sufficiently close to the cells, and the intracellular fluid is The water molecules that move from inside the cell to the outside of the cell due to the osmotic pressure difference created between the cell and the polymer solution are trapped in the water-soluble polymer matrix, further promoting the discharge of water from inside the cell. The increased concentration of proteins and sugars in the cell supercools the water inside the cell, and the vapor pressure difference between the supercooled water and the supercooled water further promotes the movement of water molecules out of the cell through the cell membrane. Prevents or suppresses freezing and the growth of ice crystals within.

On the other hand, in Comparative Example 1 in which DMSO was the test sample and Comparative Example 2 in which only the medium was used, the orthogonal projected area of the cells increased after freezing. In addition, when cryopreserving the test sample solutions of Comparative Examples 3, 4, and 6 whose intrinsic viscosity (η) is outside the range of 0.20 dL/g or more and 0.95 dL/g or less, the cells remain as they were before freezing. They had almost the same orthographic area. Furthermore, even if the intrinsic viscosity (η) is 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 deviates from the endothermic peak temperature of water, which is around 0.7°C, and - In the test sample solutions (Comparative Examples 5, 9, and 10) at temperatures below 1.4°C and above 1.1°C, sufficient cell contraction was not observed. In the cryopreservation using the test sample solutions of these comparative examples, cell dehydration did not occur or was not sufficient, and therefore, the survival rate of cells after cryopreservation was also lower than that of Example 1 in which the cells were well dehydrated. This is considered to be lower than 5. In Comparative Examples 7 and 8, the orthogonal projected area of the cells was larger than before freezing. In Comparative Example 8, it was observed that cell rupture also occurred due to freezing. In Comparative Examples 7 and 8, it is thought that the water-soluble polymer permeated into the cells. Furthermore, the cell viability of the cells after cryopreservation in Comparative Examples 7 and 8 was also low. Such cell-permeable water-soluble polymers expand and damage cells when frozen, so they are not suitable as water-soluble polymers for aqueous polymer solutions used for cryopreservation of biological samples. I can think of no. In addition, in Production Examples 1 to 3 where the intrinsic viscosity (η) was small, no change was observed in the orthogonal projected area of the cells, and it was presumed that no dehydration of the cells occurred.

In cryopreservation using the aqueous polymer solution of the present invention, damage to cells during freezing is significantly reduced due to good dehydration of cells during freezing, and this allows thawed cells to have a high survival rate. It is considered to indicate.

<Test Example 10: Evaluation of cryoprotection for various cells>
Cultured mouse-derived macrophage-like cell line (RAW264), human colon cancer-derived cells (Caco-2), and primary human lung microvascular endothelial cells (HMVEC) were each cultured at a concentration of 1×10 ⁶ cells/mL in Example 1 and Comparative Example 1 (serum-free). Thereafter, the cell suspension containing each test sample solution was frozen in a -80°C freezer in a slow cell freezer (Nalgene® Mr. Frosty). After 7 days of frozen storage, cell suspensions containing each test sample solution were taken out and rapidly thawed in a 37°C hot bath. The cell viability of the cell suspension containing each test sample solution after thawing was evaluated by trypan blue staining. The results are shown in FIG.

As shown in FIG. 9, when the aqueous polymer solution of the present invention was used for cryopreservation, all cells had a high cell survival rate that was almost equal to or higher than that of Comparative Example 1 in which DMSO was used as the test sample. Obtained. These results confirm that the aqueous polymer solution of the present invention can cryopreserve various types of cells with a high cell survival rate, regardless of whether they are primary cells or established cells, or the species of origin of the cells. It was done.

<Test Example 11: Evaluation of the effect of low molecular weight saccharides on cell viability>
Cultured primary canine mesenchymal stem cells (cyagen C160) were suspended in each test sample solution (serum-free) of Production Examples 1 to 3 at a concentration of 1×10 ⁶ cells/mL. Thereafter, the cell suspension containing each test sample solution was frozen in a -80°C freezer in a slow cell freezer (Nalgene® Mr. Frosty). After 7 days of frozen storage, cell suspensions containing each test sample solution were taken out and rapidly thawed in a 37°C hot bath. The cell viability of the cell suspension containing each test sample solution after thawing was evaluated by trypan blue staining. The results are shown in FIG. 10 and Table 1.

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

<Test Example 12: HPLC analysis of subcritically treated hyaluronic acid sample>
A 1 wt % aqueous solution of the test samples of Production Examples 1 to 3 was prepared, filtered through a 0.45 μm membrane filter (manufactured by Millipore), and the components of each test sample were analyzed by HPLC. As the mobile phase, A solution: 16mM NaH ₂ PO ₄ aqueous solution, B solution: 800mM NaH ₂ PO ₄ aqueous solution were used, and ZORBAX NH ₂ (manufactured by Agilent Technologies, Co., Ltd., column size φ4.6 x 250 mm, particle size 5 μm) was used. The components were separated using a 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 as follows: mobile phase B concentration: 0% (0 minutes) → mobile phase B concentration: 100% (60 minutes). The results are shown in Figures 11A-C. In addition, as standard samples, HA02, a disaccharide, HA04, a tetrasaccharide, HA06, a hexasaccharide, HA08, an octasaccharide, and HA10, a decasaccharide (all manufactured by Izuron) were prepared at a concentration of 0.2 wt%. Aqueous solutions containing each oligosaccharide of hyaluronic acid were prepared and subjected to HPLC analysis in the same manner. The results are shown in FIG. 11D.

Under the present HPLC analysis conditions, monosaccharides are confirmed at a retention time of 3.5 minutes, and disaccharides are confirmed at a retention time of around 9 minutes, as shown in FIG. 11D. FIG. 11A shows the analysis results of the test sample containing the cleavage product of hyaluronic acid of Production Example 1 and having a viscosity average molecular weight of 1000, and a peak corresponding to a monosaccharide was observed around the retention time of 3.5 minutes. , a peak corresponding to a disaccharide at a retention time of around 9 minutes is observed. That is, the test sample of Production Example 1 contains such monosaccharide and disaccharide components, which are thought to contribute to the effect of improving cell survival rate.

<Test Example 13: HPLC analysis of test sample of Example 1>
Regarding the test sample of Example 1 (hyaluronic acid sample with a viscosity average molecular weight of about 10,000 obtained by subcritical treatment), using the same conditions as the HPLC analysis of the test samples of Production Examples 1 to 3 above, HPLC analysis was performed. The results are shown in FIG.

Similarly, when compared with FIG. 11D, it can be seen that in the test sample of Example 1, peaks corresponding to monosaccharides and disaccharides are observed at retention times of around 3.5 minutes and around 9 minutes, respectively. That is, the test sample of Example 1 also contained such monosaccharide and disaccharide components, and it is thought that this resulted in a high cell survival rate effect.

From the above results, the polymer aqueous solution of the present invention stably vitrifies the inside of a biological sample and prevents or suppresses the growth of ice crystals within the biological sample, thereby allowing cell-permeable substances such as DMSO and ethylene glycol to It has the remarkable effect of being able to cryopreserve biological samples with a high cell survival rate without basically requiring the addition of toxic compounds and/or serum or serum-derived proteins. I know that there is. Cells are well protected and their properties are maintained.

Note that it is possible to add proteins that are not contaminated with bacteria or viruses. It is also possible to use cell-permeable and toxic compounds at low concentrations that do not impair cell function.

Claims (19)

An aqueous polymer solution containing a water-soluble polymer or its salt in an aqueous solvent and used for cryopreservation of biological samples,
The water-soluble polymer is hyaluronic acid or pullulan,
The intrinsic viscosity (η) of the polymer aqueous solution is 0. 32 dL/g or more, 0. 65 dL/g or less,
In the DSC curve of the heating process obtained under the measurement conditions (1) and (2) below using a differential scanning calorimeter (DSC),
An aqueous polymer solution characterized in that no endothermic peak is observed or the temperature at which an endothermic peak is observed is higher than -1.4°C and 1.1°C or lower when measured by DSC.
(DSC measurement conditions)
(1) After holding at 20°C for 1 minute, lower the temperature to -80°C at a cooling rate of 5°C/min.
(2) After holding at -80°C for 1 minute, raise the temperature to 20°C at a temperature increase rate of 10°C/min. In the DSC curve of the heating process obtained under the measurement conditions of (1) and (2) above,
When the endothermic peak measured by DSC is not confirmed or the temperature at which the endothermic peak is confirmed is higher than -1.4°C and 1.1°C or lower, and the aqueous polymer solution is solidified at -80°C, 2. The polymer solution according to claim 1, wherein the orthogonally projected area of the biological sample is smaller than the orthogonally projected area of the biological sample before cooling. In the DSC curve of the temperature decreasing process obtained under the measurement conditions (1) and (2) above, the calorific value of the exothermic peak in the temperature decreasing process is 0 J/g, or 65% of the corresponding calorific value of the reference solution consisting of water. % or less. The polymer solution according to any one of claims 1 to 3, wherein the exothermic peak measured by DSC has a calorific value of 0 J/g or 40% or less of the corresponding calorific value of a reference solution consisting of water. Polymer aqueous solution. The polymer aqueous solution according to any one of claims 1 to 4, wherein the polymer aqueous solution contains the water-soluble polymer or its salt in a content of 0.1 w/v% or more and 20 w/v% or less. The aqueous polymer solution according to any one of claims 1 to 5, wherein the water-soluble polymer is a water-soluble polymer containing a sugar residue. 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. The aqueous polymer 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. The aqueous polymer solution according to claim 7, wherein the biological sample is a sperm, an egg, or a fertilized egg. The aqueous polymer solution according to any one of claims 1 to 9, which is used for vitrification preservation of the biological sample. A method for preventing or suppressing the growth of ice crystals in a biological sample during cryopreservation, the method comprising contacting the biological sample with a polymer solution according to any one of claims 1 to 10. An aqueous polymer solution containing a water-soluble polymer or its salt in an aqueous solvent and used for cryopreservation of biological samples,
The water-soluble polymer is hyaluronic acid or pullulan,
The intrinsic viscosity (η) of the polymer aqueous solution is 0. 32 dL/g or more, 0. 65 dL/g or less,
In the DSC curve of the heating process obtained under the measurement conditions (1) and (2) below using a differential scanning calorimeter (DSC),
The biological sample is placed in an aqueous polymer solution characterized in that 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. a step of including;
A method for cryopreservation of a biological sample, comprising the step of cooling and solidifying the aqueous polymer solution containing the biological sample.
(DSC measurement conditions)
(1) After holding at 20°C for 1 minute, lower the temperature to -80°C at a cooling rate of 5°C/min.
(2) After holding at -80°C for 1 minute, raise the temperature to 20°C at a temperature increase rate of 10°C/min. In the DSC curve of the heating process obtained under the measurement conditions of (1) and (2) above,
The endothermic peak measured by DSC is not confirmed, or the temperature at which the endothermic peak is confirmed is higher than -1.4 °C and 1.1 °C or lower,
The method for cryopreservation of a biological sample according to claim 12, wherein the orthogonal projected area of the biological sample in a state in which the aqueous polymer solution is solidified at -80° C. is smaller than the orthogonal projected area of the biological sample before the temperature is lowered. . The method for cryopreservation of a biological sample according to claim 12 or 13, further comprising the step of preserving the aqueous polymer solution containing the biological sample at a temperature of −27° C. or lower. The cryopreservation of a biological sample according to any one of claims 12 to 14, wherein the step of cooling and solidifying the aqueous polymer solution containing the biological sample is performed by a slow freezing method at a cooling rate of 10° C./min or less. Method. In the DSC curve of the temperature decreasing process obtained under the measurement conditions (1) and (2) above, the calorific value of the exothermic peak in the temperature decreasing process is 0 J/g, or 65% of the corresponding calorific value of the reference solution consisting of water. % or less, the method for cryopreservation of a biological sample according to any one of claims 12 to 15. 17. The polymer solution according to any one of claims 12 to 16, wherein the exothermic peak measured by DSC has a calorific value of 0 J/g or 40% or less of the corresponding calorific value of a reference solution consisting of water. Cryopreservation method for biological samples. The method for cryopreservation of a biological sample according to any one of claims 12 to 17, wherein the biological sample is a cell, a tissue, or a tissue-like substance such as a membrane or an aggregate. 19. The method for cryopreservation of a biological sample according to claim 18, wherein the biological sample is a sperm, an egg, or a fertilized egg.

Images