JPWO2020166712A1 - Solidified material containing aqueous polymer solution for cryopreservation and biological sample - Google Patents

Abstract

It is an object of the present invention to provide a solidified body containing a polymer aqueous solution for cryopreservation of a biological sample and a biological sample having a high cell survival rate. An aqueous solution of a polymer containing a water-soluble polymer or a salt thereof in an aqueous solvent and used for cryopreservation of a biological sample, wherein the ultimate viscosity (η) of the aqueous solution of the polymer is 0.20 dL / g or more, 0. It is 95 dL / g or less, and the normal projection area of the biological sample in the state of being solidified at -80 ° C is reduced to less than 8/10 as compared with that before solidification, and the temperature is lowered using a differential scanning calorimeter (DSC). In the DSC curve of the process, the calorific value of the exothermic peak in the temperature lowering process is 0 J / g, or 65% or less of the corresponding calorific value of the reference solution consisting of water, and the aqueous polymer solution and freeze-solidification thereof. The body.

Description

The present invention relates to a polymer aqueous solution for cryopreservation of a biological sample and a solidified body containing the 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 in humans but also in the field of veterinary medicine. Bone marrow-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells collected from living organisms are increased in large quantities after collection and used in regenerative medicine and regenerative medicine research as described above. At this time, it is common to cryopreserve the cells that have been excessively increased and use them as appropriate. In addition, there is an increasing demand for a stable supply of such cells.

In the cell cryopreservation mechanism, when ice crystals grow inside the cell during the process of freezing and / or thawing, the cell membrane and intracellular structure are damaged, and the protein of the cell is denatured, resulting in fatal damage to the cell. It is known to receive. Therefore, it is important to prevent intracellular freezing when storing cells in a cryopreservation, and low-molecular-weight compounds such as dimethylsulfoxide (DMSO), glycerin, and propylene glycol usually permeate into the cells during cryopreservation. As a type freeze protection reagent, it is used by adding it to a buffer solution such as a culture medium (Patent Document 1). Of these, DMSO is most often used and has a good effect of protecting cells and organelles. However, in the intracellular permeation type cryopreservation solution, since the low molecular weight compound which is a cryoprotective reagent permeates into the cell, there is a concern that the cryoprotective reagent may affect the cells (Non-Patent Document 1).

Therefore, attempts have been made to use a natural freeze-protecting agent as a freeze-protecting reagent instead of a chemical substance. For example, it is known that disaccharides, oligosaccharides, or high molecular weight polysaccharides are added to a buffer solution such as a culture medium as a non-penetrating cryoprotective reagent.

In addition, a method of retaining a biological component in a crosslinked body that forms a hydrogel is also being investigated. Patent Document 2 describes a living body using 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 having a weight average molecular weight of 5000 to 4 million. Preservatives for ingredients are listed. The modified hyaluronic acid reacts with the hydroxyl group of a compound having a plurality of hydroxyl groups such as polyvinyl alcohol to form a crosslinked product in which the modified hyaluronic acid is crosslinked, and the biological component is embedded in this agar-like hydrogel to form a preservative. It is used as. The molecular weight of the hydrogel described in Patent Document 2 as an actual preservative is estimated to be several million or more. In Patent Document 2, the biological components are stored in a refrigerator at about 4 ° C., and the storage period is about several days.

Further, in Patent Document 3, a cryopreservation composition containing a carboxylated polyamino acid in which the amino group of the polyamino acid is blocked by carboxylation (or acetylation) with a carboxylic acid anhydride and an organic amphoteric agent is used. Has been described. 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 Gazette Japanese Unexamined Patent Publication No. 2012-235728

REJUVENATION RESEARCH Volume 18 Number 5 (2015): 422-436, Mary Ann Liebert, Inc.

The intracellular osmotic cryoprotectant slows the formation rate of ice crystals formed in cells by promoting the dehydration of cells, and inhibits ice crystal formation. In particular, DMSO easily penetrates into cells and is therefore effective for cryopreservation of cells having a complicated structure such as mammalian cells, but as described in Non-Patent Document 1 described above, like DMSO. Chemicals are cytotoxic. It is considered that the effect of toxicity increases as the intracellular penetration of the cryoprotectant progresses and the intracellular concentration increases.

In addition, 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 been reported to affect the differentiation of ES cells (Cryobiology. 2006 Oct; 53 (2): 194-205.). Therefore, the use of DMSO as a cryoprotectant may not be suitable for cell preservation when it is necessary to maintain undifferentiated or functional properties in stem cells. In addition, when storing samples using DMSO for a long period of time, it is indispensable to store the samples in liquid nitrogen or in an atmosphere that requires handling management, and it is considered to be an issue for the spread of regenerative medicine and regenerative medicine research. Be done.

On the other hand, natural cryoprotectants such as sugar are compatible with cells, but have a large molecular size and are difficult to be taken up into cells. Therefore, it is considered that intracellular freezing cannot be sufficiently suppressed only by adding extracellularly.

As mentioned above, the preservatives for biological components described in Patent Document 2 have a test storage period of only a few days, and a better cryopreservation technique is required for storage in units of several months. It is believed that there is. Further, in order to produce the hydrogel described in Patent Document 2, it is necessary to introduce a modifying group into hyaluronic acid, and further, to dissolve the gel for recovery of the biological component after storage from the hydrogel. The use of additives is also required. However, if such an additive is introduced into the cryopreservation method, it can be used for research and testing, but it is difficult to use it for actual medical use due to the risk of contamination with impurities.

Patent Document 3 discloses a cryopreservation composition containing a carboxylated polyamino acid in which the amino group of the polyamino acid is modified with a carboxylic acid, an organic amphoteric agent, and optionally a polysaccharide, but the cells are cryopreserved. The survival rate was not sufficient when they were allowed to do so.

In addition, the fructan described in Patent Document 4 also showed the death of cells due to rupture and the like, and the cells could not be sufficiently cryopreserved.

A method using a non-penetrating cryoprotectant reagent is also known, but such a preservation reagent alone has a low effect on cells, but a sufficient preservation effect cannot be obtained. Therefore, a compound in which the amount of the compound is reduced or substituted by combining an intracellular penetrating compound such as intracellular penetrating DMSO, glycerin, and propylene glycol with a non-penetrating substance is commercially available. However, no cryoprotectant that exhibits a good cryopreservation effect with only natural components such as biological constituents and is practical has not been reported.

The present invention has been made in view of the above problems, and provides a polymer aqueous solution for cryopreservation for appropriately preserving a biological sample. In particular, a biological sample is basically free of chemical substances such as dimethyl sulfoxide (DMSO), propylene glycol (PG), and ethylene glycol (EG), and is basically free of serum and serum-derived proteins. A polymer aqueous solution for cryopreservation that enables stable long-term storage at a temperature of -27 ° C or lower, a method for using it to prevent or suppress the growth of ice crystals in a biological sample, and a solidified substance containing the biological sample. The purpose is to provide.

The present invention is a polymer aqueous solution containing a water-soluble polymer or a salt thereof in an aqueous solvent and used for cryopreserving a biological sample, in which the ultimate viscosity (η) of the polymer aqueous solution is 0.20 dL /. The normal projection area of the biological sample in a state where the polymer aqueous solution is solidified at −80 ° C. is g or more and 0.95 dL / g or less, and the normal projection area of the biological sample in the culture solution (in the medium) before solidification is The area is reduced to less than 8/10 of the normal projection area, and the DSC in the temperature lowering process obtained by the following measurement conditions (1) and (2) using a differential scanning calorimeter (DSC) for the aqueous polymer solution. In the curve, the present invention relates to a polymer aqueous solution characterized in that the calorific value of the exothermic peak in the temperature lowering process is 0 J / g or 65% or less of the corresponding calorific value of the reference liquid consisting of water. Here, the DSC scanning is performed by (1) holding at 20 ° C. for 1 minute, then lowering the temperature to −80 ° C. at a temperature lowering rate of 5 ° C./min, (2) holding at −80 ° C. for 1 minute, and then 10 ° C./. It is carried out under the condition that the temperature is raised to 20 ° C. at a heating rate of min.

In the polymer aqueous solution of the present invention, the endothermic peak measured by DSC is not confirmed in the DSC curve of the temperature rising process obtained by the measurement conditions of (1) and (2) above using a differential scanning calorimeter (DSC). Alternatively, it is desirable that the temperature at which the endothermic peak is confirmed exceeds -1.4 ° C and is 1.1 ° C or lower.

Further, the normal projection area of the biological sample in the state where the polymer aqueous solution is solidified at −80 ° C. is 1/3 of the normal projection area of the biological sample in the culture medium (in the medium) before solidification. It is desirable that the size is 5 or more.

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

Further, it is preferable that the temperature at which the endothermic peak is confirmed is −1.3 ° C. or higher and 0.83 ° C. or lower.

In the present specification, "the endothermic peak is not confirmed" is considered to be "the endothermic peak is not confirmed" unless the endothermic peak is confirmed in the range of -30 ° C to + 5 ° C. This is because if water containing a water-soluble polymer shows an endothermic peak, it is generally in this range. In addition, the normal projection area of the biological sample and the brightness of the Munsell color system in the region occupied by the biological sample and the matrix region do not depend on the temperature after freeze-solidification, but the normal projection area of the biological sample and the living body. When the brightness of the Munsell color system in the region occupied by the sample and the matrix region is temperature-dependent, the normal projection area and the brightness of the Munsell color system are measured at −80 ° C., respectively.

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

It is desirable that the aqueous polymer solution for cryopreservation of the biological sample of the present invention does not contain dimethyl sulfoxide, which is an intracellular permeation type cryoprotectant. Further, it is desirable that the aqueous polymer solution for cryopreservation of the biological sample of the present invention does not contain a cytotoxic cryoprotectant such as ethylene glycol. This is because they are harmful to the cells after thawing.

In the polymer aqueous solution of the present invention, in the DSC curve of the temperature lowering process obtained by the measurement conditions of (1) and (2) of the above DSC, the calorific value of the exothermic peak in the temperature lowering process is 0 J / g or water. It is preferably 40% or less of the corresponding calorific value of the reference liquid composed of.

A polymer aqueous solution for cryopreserving a biological sample containing a water-soluble polymer or a salt thereof in a content of 0.1 w / v% or more and 20 w / v% or less is preferable.

A polymer aqueous solution for cryopreserving a biological sample in which the water-soluble polymer is a water-soluble polymer containing sugar residues is preferable.

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

The biological sample is preferably a polymer aqueous solution for cryopreservation of a biological sample such as mesenchymal stem cells, blood cells, endothelial cells, or a tissue for transplantation.

A polymer aqueous solution for cryopreserving a biological sample in which the biological sample is a sperm, an egg, or a fertilized egg is preferable.

It is preferable that the aqueous polymer solution for cryopreserving the biological sample of the present invention is used for vitrification storage of the biological sample.

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

The present invention is also a solidified body containing a matrix containing an aqueous solvent and a water-soluble polymer or a salt thereof and a biological sample, and the biological sample is compared with a culture solution (in a medium) before solidification. It has a normal projection area smaller than 8/10, preferably reduced to 1 / 3.5 or more, and is a mansell table of the region occupied by the biological sample when viewed from the light emitting side when visible light is transmitted. The difference between the lightness in the color system and the lightness in the Mansell color system of the region occupied by the matrix is 3 or less, and the measurement conditions of the following (1) to (2) using the differential scanning calorimeter (DSC) of the matrix. In the DSC curve of the temperature raising process obtained by the above, the heat absorption peak of the heat absorption peak in the temperature raising process is 0 J / g or 65% or less of the corresponding heat absorption amount of the reference liquid composed of water. Regarding solidified bodies. Here, the DSC scanning is performed by (1) holding at 20 ° C. for 1 minute, then lowering the temperature to −80 ° C. at a temperature lowering rate of 5 ° C./min, (2) holding at −80 ° C. for 1 minute, and then 10 ° C./. It is carried out under the condition that the temperature is raised to 20 ° C. at a heating rate of min.

It is desirable that the ultimate viscosity (η) of the aqueous polymer solution containing the aqueous solvent and the water-soluble polymer or a salt thereof before freeze-solidification is 0.20 dL / g or more and 0.95 dL / g or less.

In the DSC curve of the heating process obtained by the measurement conditions of (1) and (2) of the above DSC, the endothermic peak is not confirmed, or the temperature at which the endothermic peak is confirmed exceeds -1.4 ° C. 1 A solidified body having a temperature of 1 ° C. or lower is desirable.

Here, "the endothermic peak is not confirmed" is considered to be "the endothermic peak is not confirmed" unless the endothermic peak is confirmed in the range of -30 ° C to + 5 ° C. This is because if water containing a water-soluble polymer shows an endothermic peak, it is generally in this range.

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

It is desirable that the solidified product of the present invention does not contain dimethyl sulfoxide, which is an intracellular permeation type freeze-protecting substance for biological samples. Further, it is desirable that the solidified product of the present invention does not contain a cryoprotectant that is cytotoxic to a biological sample such as ethylene glycol. This is because they are harmful to the biological sample after thawing.

The solidified product of the present invention is preferably a solidified frozen product (frozen solidified product).

It is preferable that the solidified body of the present invention is a solidified body containing a water-soluble polymer or a salt thereof in a content of 0.1 w / v% or more and 20 w / v% or less.

In the solidified body of the present invention, in the DSC curve of the heating process obtained by the measurement conditions of (1) and (2) of the above DSC, the endothermic peak of the heat absorbing peak in the heating process is 0 J / g or. It is preferably 40% or less of the corresponding endothermic amount of the reference liquid consisting of water.

It is preferable that the biological sample contained in the solidified body is a cell, and the solidified body in which the cell is a mammalian cell is preferable.

The biological sample contained in the solidified body is a cell, and the solidified body in which the cell is a mammalian mesenchymal stem cell, a mammalian blood cell, or a mammalian endothelial cell is preferable.

The solidified product of the present invention can preferably be melted and used as a solution for administration of a biological sample. Since the solidified product of the present invention does not contain a cell-permeable and toxic compound such as DMSO or ethylene glycol and does not contain serum or serum-derived protein, it can be used as a biological sample administration solution as it is after thawing. It is possible to add proteins that are not contaminated with bacteria or viruses. It is also possible to use compounds that are cell permeable and toxic at low concentrations that do not impair cell function.

The amount of HGF produced by the biological sample contained in the solution for administration of the biological sample in which the solidified body of the present invention has been melted is such that the biological sample in the solidified body containing DMSO instead of the water-soluble polymer or a salt thereof is melted. It is preferable that it is suppressed as compared with the amount of HGF produced later.

The amount of IL-10 produced by the biological sample contained in the solution for administration of the biological sample in which the solidified body of the present invention is melted contains DMSO instead of the water-soluble polymer or a salt thereof. Is preferably increased as compared with the amount of IL-10 produced after thawing.

Further, it is desirable that the aqueous polymer solution for cryopreservation of the biological sample of the present invention and the solidified product do not contain a cell-permeable and cytotoxic compound such as dimethylsulfoxide or ethylene glycol. This is because they are toxic to cells after thawing.

In the method for cryopreserving a biological sample using the polymer aqueous solution of the present invention, the biological sample is cryopreserved including a step of holding the polymer aqueous solution containing the biological sample at a temperature of −27 ° C. or lower. The method is preferred.

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

A method for cryopreserving a biological sample in which the step of cooling and solidifying the polymer aqueous solution containing the biological sample is performed by a slow freezing method having a cooling rate of 10 ° C./or or less is preferable. Desirably, the step of cooling and solidifying the aqueous polymer solution containing the biological sample is performed by a slow freezing method having a cooling rate of 1 ° C./min or less.

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

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

According to the polymer aqueous solution and the solidified substance of the present invention, a biological sample such as a cell or a tissue without using a cell-penetrating and toxic compound such as DMSO or ethylene glycol which is an intracellular permeation type freeze-protecting substance. Can be stored properly. Furthermore, since the cryopreservation using a water-soluble polymer in the present invention is an intracellular non-penetrating type, it is less toxic to cells even though it exhibits a high cryopreservation effect.

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

The "extreme viscosity (η)" of the aqueous polymer solution containing a 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) Dissolve a predetermined amount of NaCl in ion-exchanged water at 30 ° C. to prepare a 0.2 M NaCl solution (standard solution).
(2) A sample of a water-soluble polymer or a salt thereof is dissolved in a standard solution at 30 ° C. to prepare a stock solution. When a sample of a water-soluble polymer or a salt thereof is obtained as a solution, the solid content obtained by removing the solvent from the solution is used as a sample of the water-soluble polymer or a salt thereof. In the case of a mixed sample containing a plurality of water-soluble polymers, the sample of the mixture is used as it is. Even if it is a mixture of a water-soluble polymer mixed with impurities, it can be used as a sample as it is. At this time, the viscosities of the standard solution and the undiluted solution are measured, and the relative viscosity of the undiluted solution with respect to the standard solution is adjusted to 2.0 to 2.4.
(3) The stock solution at 30 ° C. is diluted with a standard solution at 30 ° C. so as to be 5/4, 5/3, 5/2 times, respectively.
(4) Measure the viscosities of the standard solution, the undiluted solution and the diluted solution at 30 ° C., respectively. 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 undiluted solution and the diluted solution by the viscosity of the standard solution, and the reduced viscosity is derived based on the following formula.

Here, η _sp : the reduced viscosity of the water-soluble polymer or its salt [mL / g], η _r : the relative viscosity of the water-soluble polymer or its salt [-], C: the 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 (polymer or saccharide concentration = 0) of the approximate straight line is defined as the ultimate viscosity of the water-soluble polymer or a salt thereof.

As the aqueous solvent used for dissolving the water-soluble polymer or the salt thereof in the present invention, water, a physiological solution, or sodium ion or potassium ion so as to be substantially the same as the osmotic pressure of body fluid or cell fluid. , An isotonic aqueous solution in which the salt concentration, sugar concentration and the like are adjusted with calcium ions and the like is preferable. Specifically, for example, water, physiological saline, phosphate buffered saline (PBS), which is a buffered saline solution, Dulvecolinic acid buffered saline solution, Tris buffered saline solution (Tris buffered saline solution). Tris Buffered Saline (TBS), HEPES buffered saline, etc., balanced salt solution such as Hanks balanced salt solution, Ringer's solution, lactic acid Ringer's solution, acetate Ringer's solution, Ringer's bicarbonate solution, or D-MEM, E-MEM, αMEM, RPMI- 1640 medium, basal medium for animal cell culture such as Ham's F-12, Ham's F-10, M-199, general culture solution for various cells or tissues, commercially available medium and the like can be mentioned. can.

According to the polymer aqueous solution and solidified body of the present invention (frozen solidified body, hereinafter, the solidified body is referred to as “frozen solidified body”), biological samples such as cells are protected by freezing protection such as highly toxic DMSO and ethylene glycol. It can be cryopreserved without using a substance while maintaining the viability and properties of the biological sample. Since the water-soluble polymer or a salt thereof, which is a cryoprotectant for the aqueous polymer solution of the present invention and is contained as a solute in the aqueous solution of the present invention, is not permeable to cell membranes, it is a living body when vitrifying a biological sample such as a cell. It does not penetrate into the sample and swell the living tissue, but rather promotes the removal of water from the cells. In the polymer aqueous solution of the present invention, the molecular species and the degree of polymerization (molecular weight) of the polymer are adjusted, and / or by adding a low molecular weight or single molecule compound to the aqueous solution of the water-soluble polymer. The ultimate viscosity (η) of the molecular aqueous solution and the temperature and calorific value of the heat absorption peak of melting in the heating process are adjusted. In the present invention, the ultimate viscosity (η) of the aqueous polymer solution is in the range of 0.20 dL / g or more and 0.95 dL / g or less, and the calorific value of the exothermic peak measured by DSC is 0 J / g or standard water (standard water). By adjusting the calorific value of ultrapure water to 65% or less, or by adjusting the heat absorption peak to 0 J / g or 65% or less of the heat absorption of standard water (ultrapure water), the cells The cryopreservation effect of living tissues such as is improved.

The polymer aqueous solution for cryopreservation of the biological sample of the present invention is an intracellular non-penetrating cryopreservation solution, and the polymer aqueous solution and its frozen solidified substance are intracellular permeation type such as DMSO and ethylene glycol. Contains no chemicals. Therefore, damage to biological samples, cytotoxicity, and effects on cell properties as reported with respect to DMSO can be suppressed to a low level. That is, the properties of the biological sample in the biological sample during and after cryopreservation can be maintained.

Further, the polymer aqueous solution for cryopreservation of the biological sample of the present invention and the frozen solidified product thereof cryopreserve the biological sample while maintaining the viability and properties of the biological sample without using serum and / or serum-derived protein. The biological sample is not contaminated with bacteria or viruses because it is possible to do so.

Further, according to the method for cryopreserving a biological sample using a polymer aqueous solution and a frozen solidified product of the present invention, a frozen biological sample is prepared by using a chemical substance having cytotoxicity such as DMSO or ethylene glycol or a protein such as serum. It can be stably stored for a long period of time at a temperature of −27 ° C. or lower, that is, in a deep freezer or the like. It is possible to add proteins that are not contaminated with bacteria or viruses. It is also possible to use compounds that are cell permeable and toxic at low concentrations that do not impair cell function.

The range of the cryopreservation temperature 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. The lower limit is preferably -196 ° C. or higher, preferably −150 ° C. or higher.

It is a figure which shows the cell viability of the primary human mesenchymal stem cell in the cryopreservation using the test sample solution of this invention. It is a figure which shows the analysis result of the test sample solution by the differential scanning calorimetry. It is an enlarged view which shows the analysis result of the test sample solution by the differential scanning calorimetry. It is a figure which shows the analysis result of the test sample solution containing ultrapure water by the 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. It is a figure which shows the analysis result of the test sample solution containing the test sample of Example 1 by the differential scanning calorimetry. It is a figure which shows the long-term preservation effect of the primary human mesenchymal stem cell in the cryopreservation using the test sample solution of this invention. It is a figure which shows the long-term preservation effect of the primary human mesenchymal stem cell in the refrigerated storage using the test sample solution of this invention. It is a figure which shows the production amount of HGF in the primary human mesenchymal stem cell after cryopreservation using the test sample solution of this invention. It is a figure which shows the production amount of IL-10 in the primary human mesenchymal stem cell after cryopreservation using the test sample solution of this invention. It is a figure which shows the expression level of the undifferentiated biomarker in the primary human mesenchymal stem cell after cryopreservation using the test sample solution of this invention. It is a figure which shows the intracellular vitrification state after freezing in a control test (Comparative Example 2). It is a figure which shows the intracellular vitrification state after freezing using the test sample solution of the comparative example 1. FIG. It is a figure which shows the intracellular vitrification state using the test sample solution of Example 1. FIG. It is a figure which shows the difference in brightness of the intracellular vitrification state. It is a figure which quantified the difference in the lightness of the intracellular vitrification state according to the Munsell lightness (0 to 10), and obtained the difference in the lightness between the solvent and the inside of the cell. It is a figure which shows the freeze protection effect on various cells in cryopreservation using the test sample solution of this invention. It is a figure which shows the cell viability of the primary canine mesenchymal stem cell in cryopreservation using a low molecular weight saccharide. It is a figure which shows the HPLC analysis result of the test sample by various production examples. It is a figure which shows the HPLC analysis result of the test sample by various production examples. It is a figure which shows the HPLC analysis result of the test sample by various production examples. It is a figure which shows the HPLC analysis result for the calibration curve of the sugar having a predetermined number of sugars. It is a figure which shows the HPLC analysis result of the test sample which has the polymer chain of this invention.

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

The "extreme 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の極限粘度とする。Extreme viscosity measurement:
(1) Dissolve a predetermined amount of NaCl in ion-exchanged water at 30 ° C. to prepare a 0.2 M NaCl solution (standard solution).
(2) A sample of a water-soluble polymer or a salt thereof is dissolved in a standard solution at 30 ° C. to prepare a stock solution. When a sample of a water-soluble polymer or a salt thereof is obtained as a solution, the solid content obtained by removing the solvent from the solution is used as a sample of the water-soluble polymer or a salt thereof. In the case of a mixed sample containing a plurality of water-soluble polymers, the sample of the mixture is used as it is. Further, even if the water-soluble polymer contains impurities, it is used as a sample as it is. However, this does not apply when measuring the viscosity average molecular weight described later. 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 it is an unknown water-soluble polymer, the substance of the water-soluble polymer is identified by HPLC, LC-MS, LC-IR, or the like, and the viscosity average molecular weight is calculated as described later. When a plurality of unknown water-soluble polymers are contained, each component is separated and fractionated, and the substance of each water-soluble polymer is identified by HPLC, LC-MS, LC-IR, etc., as described later. Calculate the viscosity average molecular weight. 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. If impurities that affect the calculation of the viscosity average molecular weight are included, the impurities are removed or the water-soluble polymer is fractionated before measurement. The viscosities of the standard solution and the undiluted solution are measured, and the relative viscosity of the undiluted solution with respect to the standard solution is adjusted to 2.0 to 2.4.
(3) The stock solution at 30 ° C. is diluted with a standard solution at 30 ° C. so as to be 5/4, 5/3, 5/2 times, respectively.
(4) Measure the viscosities of the standard solution, the undiluted solution and the diluted solution at 30 ° C., respectively. 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 undiluted solution and the diluted solution by the viscosity of the standard solution, and the reduced viscosity is derived based on the following formula.

Here, η _sp : the reduced viscosity of the water-soluble polymer or its salt [mL / g], η _r : the relative viscosity of the water-soluble polymer or its salt [-], C: the 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 (polymer or saccharide concentration = 0) of the approximate straight line is defined as the ultimate viscosity of the water-soluble polymer or a salt thereof.

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 ultimate 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 is formed by polymer chains in the cooling process. Aqueous solvent molecules can be well trapped in the resulting matrix. This allows the water to be solidified and / or frozen in a vitrified state without crystallizing by limiting the molecular movement of the water in the aqueous solvent during cooling. Usually, in the freezing method called the vitrification method, when the solution freezes, the solute (freeze protective agent for preventing frost damage) is removed from the crystals, so that the salt concentration in the residual solution increases, and the salt concentration in the residual solution increases inside and outside the cell. It is a method of dehydrating the inside of cells by causing an osmotic pressure difference and vitrifying the inside of cells, and is applied to cells having a particularly low survival rate after thawing. In such a method, in order to make water easier to vitrify, the concentration of the solute (freeze protective agent) is increased and the cooling rate is increased. However, it has been known that when the osmotic pressure difference is increased, the damage to the cells is also increased, and the problem that recrystallization occurs at the time of lysis and the cells are damaged. In addition, there are technical difficulties.

According to the aqueous polymer solution of the present invention, when used as a cryopreservation solution for a biological sample such as cells, the inside of the biological sample is dehydrated by the action of the polymer chains of the water-soluble polymer dissolved in the aqueous polymer solution. Since the inside of the biological sample is also vitrified, the formation of ice crystals in the biological sample is suppressed or prevented, and the osmotic pressure shock in the biological sample at the time of freezing, which is a problem in the conventional vitrification method, is weakened. be able to. Therefore, it is not necessary to contain chemical substances such as dimethyl sulfoxide (DMSO) and ethylene glycol which are cytotoxic, and the aqueous polymer solution of the present invention does not contain DMSO and / or ethylene glycol.

In the aqueous polymer solution containing a water-soluble polymer in the present invention, water molecules of the solvent are present in the network matrix of the polymer at the time of freezing, and free water or bound water (a state in which water molecules are entangled and trapped by the polymer). It is considered that it exists in the state of. Further, it is considered that the water molecules excluded from the inside of cells or the like due to the difference in osmotic pressure are replaced with the macromolecules around the biological sample such as cells. In order to realize this state, it is necessary to adjust the ultimate viscosity of the aqueous polymer solution to 0.25 to 0.95 dL / g. If the extreme viscosity of the aqueous polymer solution is too small, the polymer will diffuse and water molecules cannot be entangled and captured in the network of water-soluble polymers. On the other hand, if the extreme viscosity of the aqueous polymer solution is too high, the substitution with water molecules in the vicinity of the biological sample does not proceed. In addition, there may be a problem that the handling property is deteriorated when the biological sample is contained in the aqueous polymer solution for cryopreserving the biological sample. By setting the ultimate viscosity of the aqueous polymer solution in the range of 0.20 dL / g or more and 0.95 dL / g or less, when the aqueous polymer solution of the present invention is cooled, it is in an amorphous glass state in a frozen state. Is stabilized. Therefore, the cells are not easily damaged by cooling and freezing, and the cells can be stably and efficiently cryopreserved. Cell destruction due to the formation of ice crystals is also unlikely to occur. Therefore, the survival rate of cells in the biological sample after thawing the biological sample after cryopreservation of the present invention is high. For example, the ultimate viscosity (η) of the aqueous polymer solution may be preferably 0.32 dL / g or more and 0.65 dL / g or less.

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

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

The monomer having a hydrophilic group is, for example, a sugar residue. In this case, the polymer in the present invention may be a water-soluble polymer containing a polymer in which sugar residues are bonded by a glycosidic bond as a repeating unit and derivatives thereof. As the sugar residue, a monosaccharide or a monosaccharide in which the hydroxyl group and / or hydroxymethyl group of the monosaccharide is substituted, for example, a hydroxyl group and / or a hydroxymethyl group is a carboxyl group, an amino group, or an N-acetyl. Examples thereof include, but are not limited to, monosaccharides substituted with at least one substituent selected from the group consisting of an amino group, a sulfooxy group, a methoxycarbonyl group and a carboxymethyl group.

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

For example, examples of the monosaccharide substituted with a carboxyl group include uronic acid. Examples of uronic acid include glucuronic acid, iduronic acid, mannuronic acid and galacturonic acid. Examples of the monosaccharide substituted with an amino group include amino sugars. Examples of amino sugars include glucosamine, galactosamine, mannosamine and muramic acid. Examples of the monosaccharide substituted with the N-acetylamino group include N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine and N-acetylmuramic acid. Examples of the monosaccharide substituted with the sulfooxy group include galactose-3-sulfuric acid and the like. Examples of monosaccharides having a plurality of substituents include N-acetylglucosamine-4-sulfate, iduronic acid-2-sulfate, glucuronic acid-2-sulfate, N-acetylgalactosamine-4-sulfate, neuraminic acid and N. -Acetylneuraminic acid and the like.

For example, the water-soluble polymer in the present invention is a polymer containing a monosaccharide as a repeating unit as described above. For example, the water-soluble polymer in the present invention may be a polymer containing 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 contain a six-membered ring structure in its main chain. This is because polysaccharides and the like having a six-membered ring structure in the main chain may easily form a polymer network structure around cells via hydrogen bonds. Further, 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 glycosaminoglycan. Further, it may be a sulfated polysaccharide in which one or more hydroxyl groups are substituted with a sulfooxy group. Examples of the polymer that can be used in the present invention include, but are not limited to, hyaluronic acid, dextran, pullulan, chondroitin sulfate, and the like.

The water-soluble polymer or salt thereof used in the present invention may be of natural origin or may be chemically synthesized. A commercially available water-soluble polymer or a salt thereof may be used as it is. Using a naturally-derived polymer compound having a larger molecular weight or a commercially available polymer compound, 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 be a water-soluble polymer in the invention. Further, each monomer may be of natural origin, may be used by modifying or substituting a naturally derived monomer, or may be chemically synthesized. For example, preferably, the monomer contained in the water-soluble polymer in the present invention is a biological constituent. It is considered that the cytotoxicity is low when the aqueous polymer solution containing the water-soluble polymer is used as a cryopreservation solution for biological samples. In addition, the process required for the conventional cryopreservation solution, such as washing and removing the cryoprotectant contained in the cryopreservation solution from the biological sample after thawing, may be unnecessary or simplified.

The water-soluble polymer or salt thereof in the present invention has, for example, a viscosity average molecular weight of more than 3000 and not more than 500,000. By using a water-soluble polymer having a viscosity average molecular weight of this degree or a salt thereof, the amorphous glass state of the solvent in the frozen state is stabilized when the aqueous solution is cooled. Therefore, the cells are not easily damaged by cooling and freezing, and the cells can be stably and efficiently cryopreserved. Therefore, the survival rate of cells in the biological sample after thawing the biological sample after cryopreservation is high. When the viscosity average molecular weight of the water-soluble polymer is 3000 or less, vitrification may not occur well. Further, when the viscosity average molecular weight of the water-soluble polymer is larger than 500,000, there may be a problem that the viscosity is remarkably increased, the solubility is lowered, or the prepared solution is foamed and the handleability is deteriorated. The viscosity average molecular weight is preferably 10,000 or more, for example. 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 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 calculation formula using the ultimate viscosity (η) calculated from the above method and calculation formula. Demanded 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（レバン型）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 ultimate viscosity.

The viscosity average molecular weight M is obtained from the limit viscosity derived by the measurement and the values of K and α published in the literature and the like according to the above Mark Hoing Sakurada's formula. 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 , α = 0.5 from the literature values, and in the case of dextran, K = 6.3 × 10 ^-8 , α = 1.4, and chondroitin sulfate. Then, K = 5.8 × 10 ^-4 , α = 0.74, for fructan (levan type), K = 1.6 × 10 ^-3 , α = 0.45, and for carboxylated polylysine, K = 2.78. × 10 ^-5 , α = 0.87.

K and α are numerical values that fluctuate depending on the type of polymer. For example, the values of K and α are disclosed and published in many public documents such as "Polymer Material Handbook" (edited by the Japan Society of Polymer Science). The viscosity average molecular weight can be calculated from the ultimate viscosity (η) using the above values.

In the present invention, the molecular weight calculated by this method is defined as the viscosity average molecular weight. In the case of monosaccharides, disaccharides, and compounds considered to be monomolecules such as sucrose and glucuronic acid, the molecular weight is clearly specified from the structural formula, so the molecular weight specified from the structural formula is used as the viscosity average molecular weight. And treat it.

The hydrophilic group of the water-soluble polymer or salt thereof used in the present invention is not modified or even if modified, 50% or less of the total number of hydrophilic groups, that is, a substituent is introduced into the polymer chain. It is desirable that the number of hydrophilic groups is 50% or less of the total number of hydrophilic groups, even if they are not or have been introduced. Since it is presumed that the hydrophilic groups of the water-soluble polymer, especially the OH group, NH ₂ group, and COOH group, contribute to the protection of the biological sample, the vitrification of the solvent, and the replacement with the water molecules around the biological sample. This is because it is advantageous to improve the viability of the biological sample if these functional groups are not modified. Furthermore, when an attempt is made to introduce a functional group into a water-soluble polymer, the cryoprotective effect of the polymer may be adversely affected by the reagent used when modifying the hydrophilic group of the polymer, or a living body for regenerative medicine purposes. It may become unusable as a cryopreservation solution for cryopreservation of samples.

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

As the aqueous solvent for the aqueous solution of the polymer of the present invention, the salt concentration and sugar may be adjusted to be substantially the same as the osmotic pressure of water, a physiological solution, or a body fluid or a cell fluid by using sodium ion, potassium ion, calcium ion or the like. An isotonic aqueous solution whose concentration and the like are adjusted is preferable. Specifically, for example, water, physiological saline, phosphate buffered saline (PBS), which is a physiological saline having a buffering effect, darbecolinic acid buffered saline, and Tris buffered saline (Tris buffered saline (PBS)). Examples thereof include, but are not limited to, equilibrium salt solutions such as Tris Buffered Saline (TBS), HEPES buffered saline, Hanks equilibrium salt solution, Ringer's solution, lactic acid Ringer's solution, acetate Ringer's solution, and dicarbonate Ringer's solution. In addition, 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, the term "arbitrary component" means a component that may or may not be contained. For example, the solvent may be a 5% aqueous glucose solution or the like. Further, a medium for cell culture may be used as an aqueous solvent for preparing the aqueous polymer solution of the present invention. The culture medium is not particularly limited, and for example, commercially available medium, D-MEM, E-MEM, αMEM, RPMI-1640 medium, Ham's F-12, Ham's F-10, M-199, etc. Basis medium for animal cell culture, general culture medium for various cells or tissues can be exemplified. Therefore, when the biological sample is cryopreserved using the aqueous polymer solution of the present invention, the biological sample such as cells after culturing may be suspended in the aqueous polymer solution of the present invention and cryopreserved. A cryopreservation step may be performed by adding the water-soluble polymer or a salt thereof in the present invention to a culture solution containing a biological sample such as a cell culture solution or a cell suspension after culture at a desired concentration.

Further, the pH of the aqueous polymer solution of the present invention can be adjusted as needed. For example, when the hydrophilic group of the monomer having a hydrophilic group is a carboxylic acid group or the like, the aqueous polymer solution containing the water-soluble polymer on which such a monomer is polymerized may be acidic. In such a case, it is considered that by adjusting the pH to make the aqueous polymer solution a neutral solution, the solution can be more suitable for the survival of the cells to be cryopreserved, and the survival rate of the cells is improved. The salt used for pH adjustment is not limited to that, and those usually used for pH adjustment of an aqueous solution can be used.

In the polymer aqueous solution of the present invention, according to the result 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 is the following (1) to (2). In the DSC curve of the temperature lowering process obtained by the measurement conditions, the calorific value of the exothermic peak in the temperature lowering process is 0 J / g, or 65% or less of the corresponding calorific value of the reference solution consisting of water, preferably water. It is 40% or less of the corresponding calorific value of the reference liquid composed of.
DSC measurement conditions:
(1) After holding at 20 ° C for 1 minute, the temperature drops to -80 ° C at a temperature lowering rate of 5 ° C / min, and (2) after holding at -80 ° C for 1 minute, the temperature rises to 20 ° C at a heating rate of 10 ° C / min. Temperature rising.

The fact that the calorific value of the exothermic peak in the temperature lowering process in the DSC measurement is smaller (less than 100%) than the corresponding calorific value of the reference liquid consisting of water in the polymer aqueous solution of the present invention means that the frozen polymer aqueous solution is used. It means that bound water and free water that do not form ice crystals remain. That is, in the aqueous polymer solution of the present invention, the substitution of water with a water-soluble polymer and / or a low molecular weight saccharide proceeds, and water molecules are trapped in the matrix of the water-soluble polymer to cause molecular movement of water. It is restricted. As a result, dehydration of water from the inside of the cell is promoted, and water can be solidified in a glass state without crystallizing. Good vitrification in the aqueous polymer solution and the biological sample contained therein is achieved. Therefore, cryopreservation of a biological sample using the aqueous polymer solution of the present invention can suppress or prevent cell destruction due to the formation of ice crystals. Further, the aqueous polymer solution of the present invention can not only suppress or prevent ice crystal formation in the cooling process, but also suppress the recrystallization of water in the subsequent temperature raising process, that is, at the time of melting. That is, it is considered that the polymer aqueous solution of the present invention stabilizes the glass state of the biological sample in the frozen state. Since the glass state is more stabilized in this way, the aqueous polymer solution of the present invention does not require the addition of human or bovine serum or protein components of serum-derived components (eg albumin), and has high cytoprotection. Show the effect. Therefore, there is no concern about infectious diseases, and it is considered that the lots are not affected by the biologics. It is possible to add proteins that are not contaminated with bacteria or viruses.

Further, in the solidified body (frozen solidified body) of the present invention, according to the result of thermal analysis of the matrix of the frozen solidified body of the present invention by differential scanning calorimetry (DSC), the matrix contained in the frozen solidified body of the present invention. In the DSC curve of the heating process obtained by the following measurement conditions (1) and (2), the heat absorption peak of the heat absorbing peak in the heating process is 0 J / g or a reference consisting of water. 65% or less of the corresponding heat absorption of the liquid, preferably 40% or less of the corresponding heat absorption of the reference liquid consisting of water.
DSC measurement conditions:
(1) After holding at 20 ° C for 1 minute, the temperature drops to -80 ° C at a temperature lowering rate of 5 ° C / min, and (2) after holding at -80 ° C for 1 minute, the temperature rises to 20 ° C at a heating rate of 10 ° C / min. Temperature rising.

The fact that the endothermic amount of the endothermic peak in the heating process in the DSC measurement is smaller (less than 100%) than the corresponding endothermic amount of the reference liquid consisting of water in the matrix of the present invention means that ice crystals in the frozen matrix. It means that bound water and free water that do not form the water remain. That is, in the aqueous solution of the water-soluble polymer forming the matrix of the present invention, the substitution of the water-soluble polymer and / or the saccharide having a low molecular weight with water proceeds, and the water molecule is trapped in the matrix of the water-soluble polymer. It limits the molecular movement of water. As a result, dehydration of water from the cells of the biological sample contained in the matrix is promoted, and the water can be solidified in a glass state without crystallizing. Good vitrification of the matrix inside the frozen solidified body and the inside of the biological sample contained in the frozen solidified body is achieved. Therefore, in the frozen solidified product of the present invention, cell destruction due to the formation of ice crystals can be suppressed or prevented. Further, in the frozen solidified body of the present invention, not only the formation of ice crystals can be suppressed or prevented in the cooling process, but also the recrystallization of water in the subsequent temperature raising process, that is, at the time of thawing can be suppressed. That is, in the frozen solidified body of the present invention, it is considered that the glass state of the biological sample contained in the frozen solidified body in the frozen state is stabilized. Since the glass state is more stabilized in this way, the biological sample stored in the frozen solidified body of the present invention requires the addition of human or bovine serum or a protein component of serum-derived components (for example, albumin). It shows a high cytoprotective effect. Therefore, it is considered that the biological sample after thawing does not have to worry about infectious diseases and is not affected by the lot-to-lot disparity due to the biological product. It is possible to add proteins that are not contaminated with bacteria or viruses.

The calorific value and heat absorption are adjusted by selecting the molecular species of the water-soluble polymer and / or adjusting the addition amount. For example, hyaluronic acid has a small calorific value and heat absorption, but pullulan has a large calorific value and heat absorption. Further, when the content of the water-soluble polymer is increased, both the calorific value and the heat absorption amount decrease.

The polymer aqueous solution for cryopreservation of the biological sample of the present invention is subjected to DSC in the DSC curve of the temperature rising process obtained by the following measurement conditions (1) and (2) using a differential scanning calorimeter (DSC). It is desirable that 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, the temperature drops to -80 ° C at a temperature lowering rate of 5 ° C / min, and (2) after holding at -80 ° C for 1 minute, the temperature rises to 20 ° C at a heating rate of 10 ° C / min. Temperature rising.

That is, in the aqueous polymer solution of the present invention, the endothermic peak presumed to be caused by the melting of water is not observed in the process of raising the temperature of the aqueous polymer solution, or if it is observed, it is -1.4 ° C. It exists in the range of 1.1 ° C. or lower, that is, near the temperature (0.7 ° C.) at which the endothermic peak is observed by DSC measurement under the same conditions in ultrapure water. For vitrification in biological samples such as solvents and cells, it generally seems desirable to use a cryopreservation solution with a lower melting point (freezing point). However, the present inventors have found that it is desirable that the cryopreservation solution has the opposite characteristics of this technical common sense for good vitrification of the biological sample at the time of freezing. Normally, the phenomenon that the melting point (freezing point) is lower than that of water occurs when the solute is dispersed in the matrix of water, but in the aqueous polymer solution of the present invention, on the contrary, it is water-soluble. Water molecules are present in the polymer network matrix in an aqueous solvent formed of a polymer, and water exists as free water in a state where it does not freeze in the matrix, or water is a polymer even if it freezes. It freezes in the state of being caught in the water (bound water). Therefore, in the polymer aqueous solution of the present invention, it is presumed that the endothermic peak of water melting is not observed in the heating process, or the endothermic peak of pure water is observed at a temperature close to the observed temperature.

Furthermore, the present inventors, in the present invention, adjust the heat absorption peak in the polymer aqueous solution containing the water-soluble polymer or a salt thereof and the frozen solidified product in the saccharide having a lower molecular weight than the water-soluble polymer. Or it has been found that it can be done by adding the salt thereof.

The low molecular weight saccharides or salts thereof are retained in the polymer matrix by hydrogen bonds by the hydrophilic groups in the water-soluble polymer of the present invention. It is considered that the presence of a polymer holding a low molecular weight saccharide or a salt thereof around a biological sample such as a cell replaces the water molecule near the cell membrane with the low molecular weight saccharide. Therefore, it is considered that the water discharged from the biological sample such as cells to the outside of the biological sample due to the osmotic pressure difference during freezing replaces not only the macromolecules around the biological sample such as cells but also low molecular weight saccharides. Therefore, it is believed that the addition of low molecular weight saccharides or salts thereof aids in the drainage of water from within the biological sample and, by substitution with water molecules, suppresses or prevents the formation and growth of ice crystals near the cell membrane. By setting the ultimate 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 with low molecular weight saccharides can be promoted. As a result, cell membrane damage during freezing can be significantly suppressed. In other words, the low molecular weight saccharides or salts thereof can function as components for cell protection in the aqueous polymer solution and the frozen solidified product of the present invention. If the ultimate viscosity of the aqueous polymer solution of the present invention is too small, low molecular weight saccharides will diffuse into the aqueous polymer solution, and water molecules cannot be efficiently entangled and captured in the network of water-soluble polymers. On the other hand, if the ultimate viscosity of the aqueous polymer solution is too high, it is considered that the substitution of low molecular weight saccharides with water molecules does not proceed efficiently.

Since the discharge of water from the biological sample is promoted and the cells are protected in this way, living organisms such as cells frozen in the polymer aqueous solution of the present invention (that is, frozen in the frozen solidified body). The volume of the sample shrinks as compared with the biological sample before freezing. For example, the normal projection area of the biological sample in the state of being solidified at −80 ° C. in the polymer aqueous solution of the present invention is compared with the normal projection area of the biological sample in the culture medium (in the medium) before lowering the temperature (before freezing). , 1 / 3.5 or more and less than 8/10. In the freezing of the biological sample with the aqueous polymer solution of the present invention, the intracellular cells are well dehydrated at the time of freezing. This significantly reduces damage to cells during freezing, and thawed cells show high viability.

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

In order to retain low molecular weight saccharides or salts thereof in the matrix, the hydrophilic groups of the water-soluble polymers or salts thereof used in the present invention are either unmodified or hydrophilic even if modified. It is desirable that 50% or less of the total number of groups, that is, 50% or less of the total number of hydrophilic groups even if no substituent is introduced into the polymer chain. If the hydrophilic group of the water-soluble polymer is modified, the retention effect of the low molecular weight saccharide or its salt by the hydrophilic group is reduced, and the survival of the biological sample even if the low molecular weight saccharide coexists. It may not be possible to contribute sufficiently to improving the rate. Therefore, for example, _{it may not be preferable to modify the OH group or NH 2} group of the water-soluble polymer with a carboxylic acid or the like.

The low molecular weight saccharide or salt thereof in the present invention is, for example, a saccharide having a viscosity average molecular weight of 3000 or less or a salt thereof. Preferably, it may be a monosaccharide, a disaccharide, an oligosaccharide or the like 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 described above as the monomer constituting the water-soluble polymer in the present invention. For example, low molecular weight saccharides are glucose, fructose, galactose or uronic acid obtained by oxidizing the alcohol group thereof or amino sugar in which the alcohol group is replaced with an amino group, sucrose, trehalose, or a polymer or a combination thereof. .. The low molecular weight saccharide may also be a fragment of a macromolecule such as hyaluronic acid, dextran, pullulan, or chondroitin sulfate. Although not particularly limited as long as the effect of the present invention is not impaired, the low molecular weight saccharide may be, for example, a cleavage product (fragment) of glycosaminoglycan, that is, a monosaccharide constituting glycosaminoglycan, a disaccharide thereof, or a disaccharide thereof. It can be that oligosaccharide. In the case of hyaluronic acid, it is desirable to use one having 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 composed of them, 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.

The "cut product" in the present invention is a compound having a molecular weight smaller than that of the original polymer, which is considered to be obtained when the polymer is subjected to hydrolysis, enzymatic treatment, subcritical treatment, or the like. means. For example, the polymer used in the present invention may be a water-soluble polymer obtained by treatment with a larger polymer compound as described above, and the low molecular weight saccharide used in the present invention may be. , It may be a low molecular weight saccharide obtained by treatment with a water-soluble polymer. In the present invention, the cleavage product can be a monomer and / or a polymer having various degrees of polymerization of the monomer and / or a mixture thereof, which are constituents of the original polymer.

The "subcritical treatment" means that the subcritical fluid as the extraction solvent brought into the subcritical state under the conditions of a predetermined temperature and a predetermined pressure is brought into contact with the raw material to be extracted. For example, water exhibits a state of being neither a liquid nor a gas when the pressure is increased to 22.12 MPa or higher and the temperature is raised to 374.15 ° C. or higher. This point is called the critical point of water, and hot water with a temperature and pressure near the critical point is called subcritical water. By using the hydrolysis action of this sub-critical water, a desired component can be obtained from the raw material to be extracted. In the present invention, the conditions for the subcritical treatment are, for example, 150 ° C. or higher and 350 ° C. or lower, and the subcritical treatment pressure can be equal to or higher than the saturated vapor pressure at each temperature, for example, 0. It can be 5.5 MPa or more and 25 MPa or less. After the subcritical treatment, components having a predetermined molecular weight or less are separated and recovered, and can be used as a cleavage product in the present invention. Further, the hydrolysis and the enzyme treatment 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 saccharides in the present invention may be obtained simultaneously by a single subcritical treatment. That is, the water-soluble polymer and low molecular weight saccharide used in the present invention have a first molecular weight distribution in a molecular weight range of 500,000 or less, which is larger than 3000 as a viscosity average molecular weight, and have a viscosity average molecular weight of 3000 or less. It may be a subcritical treated product of a high molecular weight compound having a second molecular weight distribution in the range of molecular weight. Therefore, for example, in the method for producing an aqueous polymer solution of the present invention, a polymer, which is a polysaccharide having a molecular weight of more than 500,000 as a viscosity average molecular weight, is dissolved in water and then an extraction treatment is performed under subcritical conditions of water. Thereby, the step of obtaining the water-soluble polymer and the low molecular weight saccharide in the present invention may be included. However, of course, the water-soluble polymer and low molecular weight saccharides in the present invention may be used in combination of those separated by treatment and / or molecular weight in separate treatment steps.

The endothermic peak of the low molecular weight saccharide or salt thereof in the present invention is observed in pure water at a temperature lower than the temperature at which the endothermic peak is observed by DSC measurement (0.7 ° C.). For example, hyaluronic acid having 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 of 0.7 ° C.

On the other hand, the water-soluble polymer or salt thereof in the present invention is, for example, hyaluronic acid having a viscosity average molecular weight of 15,000, a heat absorption peak temperature of −1.4 ° C., and pullulan having a viscosity average molecular weight of 373000 at 1.49 ° C. be. When a low molecular weight saccharide or a salt thereof is combined with such a water-soluble polymer or a salt thereof, the endothermic peak temperature of the aqueous polymer solution is brought close to the endothermic peak temperature of pure water, or the endothermic peak itself is measured by DSC. Will not be observed at. For example, when hyaluronic acid having a viscosity average molecular weight of 1000 or less is added to hyaluronic acid having a viscosity average molecular weight of 15,000, the heat absorption peak temperature does not decrease but increases to −0.4 ° C. On the other hand, when hyaluronic acid having a viscosity average molecular weight of 1000 or less is added to pullulan having a viscosity average molecular weight of 373000, the heat absorption peak temperature drops to 0.83 ° C.

That is, when hyaluronic acid having a viscosity average molecular weight of 1000 or less is mixed with a water-soluble polymer whose endothermic peak temperature is lower than the endothermic peak temperature of pure water, can the endothermic peak temperature rise and approach the endothermic peak temperature of pure water? , Or can be adjusted so that the endothermic peak itself does not appear in the DSC. Further, when hyaluronic acid having a viscosity average molecular weight of 1000 or less is mixed with a water-soluble polymer whose endothermic peak temperature is higher than the endothermic peak temperature of pure water, the endothermic peak temperature is lowered and brought closer to the endothermic peak temperature of pure water. Or it is adjusted so that the endothermic peak itself does not appear in the 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 close to the endothermic peak temperature of pure water of 0.7 ° C.

The low molecular weight saccharide or a salt thereof having such an effect 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, other saccharides such as sucrose and trehalose have the same effect even if they are not hyaluronic acid. The low molecular weight saccharides used in the present invention are present between water molecules to inhibit the ice crystallization of water, or cooperate with a network of water-soluble polymers to trap water in the network. (In either water or free water), it acts to inhibit the ice crystallization of water in the first place so that the aqueous polymer solution and the frozen solidified product of the present invention can realize behavior close to that of pure water. It is presumed that it is doing.

Since the aqueous polymer solution and the frozen solidified product of the present invention do not contain osmotic compounds in the cells, it is considered that the cytotoxicity is low when they are contacted with the biological sample for cryopreservation of the biological sample. Furthermore, it is considered that the damage to cells is low because it is not necessary to increase the concentration of solute to limit the molecular motion of water. In addition, the added low molecular weight saccharides or salts thereof have a cytoprotective effect of rapidly replacing the water discharged from the cells around the cells during freezing, so that the properties of the cells must be changed during cryopreservation. Conceivable. Therefore, according to the aqueous polymer solution and the frozen solidified product of the present invention, it is considered that the biological sample can be cryopreserved while maintaining the characteristics of the cells.

Further, unlike the conventional vitrification method, the polymer aqueous solution and the frozen solidified product of the present invention do not require a large cooling rate for reducing osmotic shock during cooling. Therefore, according to the aqueous polymer solution and the frozen solidified product of the present invention, the biological sample can be efficiently cryopreserved by a simple method with reduced toxicity, and the cells thawed after the cryopreservation have high survival. Shows the rate.

The aqueous polymer solution and the frozen solidified product of the present invention contain a water-soluble polymer or a salt thereof at a concentration of about 0.1 w / v% or more and 20 w / v% or less. If the concentration of the water-soluble polymer is too low, it may not be possible to satisfactorily vitrify the solvent portion. For example, the concentration of the water-soluble polymer or a salt thereof is preferably 1 w / v% or more, and particularly preferably 5 w / v% or more. Further, if the concentration is higher than 20 w / v%, the viscosity becomes too high and the handleability may be deteriorated. Preferably, the concentration of the polymer or a salt thereof is 5 w / v% or more and 20 w / v% or less.

In the aqueous polymer solution and the frozen solidified product of the present invention, the content ratio of the low molecular weight saccharide or a salt thereof in the aqueous polymer solution of the water-soluble polymer and the low molecular weight saccharide is about 10: 1. It is preferable to include the above. If the concentration of the saccharide or its salt is too low, the effect of cell protection by the low molecular weight saccharide may not be sufficiently obtained. Further, even if the saccharide is added at a concentration of 1/10 times or more that of the water-soluble polymer, it is difficult to obtain a further effect as a cytoprotective component.

According to the results of thermal analysis of the polymer aqueous solution and the frozen solidified body of the present invention by differential scanning calorimetry (DSC), the polymer aqueous solution and the frozen solidified body of the present invention have a glass transition temperature around -23 ° C ± 4 ° C. It is desirable to have. In the aqueous polymer solution and the frozen solidified product of the present invention, as described above, vitrification is realized by entwining and trapping water molecules in the network of water-soluble polymers at the time of freezing. Therefore, it does not require a large cooling rate for cell protection during cooling as required by conventional vitrification methods. Therefore, after the biological sample is contained in the polymer aqueous solution of the present invention, it is cooled to −27 ° C. or lower, which is the glass transition temperature of the polymer aqueous solution of the present invention, to obtain a frozen solidified body. For example, simply by placing the polymer aqueous solution of the present invention containing a biological sample in a freezing container or the like and leaving it in a deep freezer at -80 ° C, the cells and tissues to be preserved are stably frozen and preserved. be able to. Since the low temperature of −150 ° C., which is required for cryopreservation of ordinary cells and the like, is not always necessary, it is possible to eliminate the need for a special container for cryopreservation of liquid nitrogen or preparation of liquid nitrogen. Therefore, the operation related to the cryopreservation of the biological sample can be remarkably simplified. Further, since the polymer aqueous solution and the frozen solidified product of the present invention can also suppress the recrystallization of water at the time of thawing, the polymer aqueous solution of the present invention can be used to freeze, store, and thaw a biological sample. A series of steps can be easily and efficiently performed without requiring a special procedure. Of course, it is also possible to cryopreserve the biological sample in liquid nitrogen or liquid nitrogen vapor using the polymer aqueous solution of the present invention and using liquid nitrogen.

Since the polymer aqueous solution and the freeze-solidified body of the present invention function as a non-penetrating cryopreservation solution and a freeze-solidified body obtained by freezing the cryopreservation solution, the polymer aqueous solution and the freeze-solidified body of the present invention are used for freezing. The biological sample to be stored is not particularly limited. It can be used for cryopreservation of various types of cells. Further, the origin of the cell is not particularly limited. Since the polymer aqueous solution and the frozen solidified product of the present invention can effectively suppress or prevent ice crystal formation and recrystallization during freezing and thawing, they are also satisfactorily used for mammalian cells having a complicated structure and the like. Can be done. Therefore, it can be applied to cryopreservation of cells of various kinds of animal species, such as mice, dogs, and humans. Furthermore, it is known that the polymer aqueous solution and the frozen solidified product of the present invention have a greater damage during freezing than general cells for culture, and the so-called conventional slow freezing method significantly reduces the survival rate. It can be particularly preferably used for cryopreservation of unavoidable stem cells, early embryos and eggs, sperms, fertilized eggs and other germ cells. Further, since the polymer aqueous solution and the frozen solidified product of the present invention do not contain chemical substances such as DMSO and ethylene glycol that can act as differentiation factors, they can be used for preservation of cells that need to be maintained undifferentiated, for example. Stem cells for regenerative medicine can also be cryopreserved without the risk of differentiation.

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

Further, the polymer aqueous solution and the frozen solidified product of the present invention are excellent in the effect of stabilizing the glass state in the frozen state, so that it is known that storage is difficult for eggs and the like having a large cell size. It can also be used for cryopreservation of cells and fertilized eggs, and organized cell structures, tissues and tissue-like substances such as tissues obtained by regenerative medicine.

That is, the aqueous polymer solution and the frozen solidified product of the present invention can be used for a biological sample selected from cells, tissues, or tissue-like substances that are membranes or aggregates, and can achieve a high survival rate. .. In particular, the high molecular weight aqueous solution and the frozen solidified product of the present invention are not related to primary cells or established cells, such as mesenchymal stem cells; hematopoietic stem cells; neural stem cells; somatic stem cells such as bone marrow stem cells and germ stem cells; blood cell cells. Advantageous in cryopreservation of endothelial cells, etc., especially in cryopreservation of primate stem cells, which are considered to be less freeze-tolerant than mice, cryopreservation of transplant tissue, and cryopreservation of germ cells in reproductive medicine. Can be used.

Further, if, for example, a water-soluble polymer which is a biological component is used as the water-soluble polymer in the aqueous polymer solution and the frozen solution of the present invention, the cryopreserved biological sample is thawed and used as it is for cell administration. It can also be used as a solution. Further, in the case of a tissue or a tissue-like substance, it is also possible to add such a polymer aqueous solution of the present invention at the stage of organization and freeze and store it as it is. This is expected to reduce post-transplant problems. In such cases, the preferred water-soluble polymer for the aqueous polymer solution and frozen solidified product of the present invention is hyaluronic acid. In particular, hyaluronic acid has a viscosity average molecular weight greater than 3000, more preferably greater than 5000, and less than 60,000, more preferably less than 20000.

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

The polymer aqueous solution of the present invention used in the cryopreservation method using the polymer aqueous solution of the present invention contains a water-soluble polymer or a salt thereof, and is in the range of 0.20 dL / g or more and 0.95 dL / g or less. The heat absorption peak measured by DSC is confirmed in the DSC curve of the heating process obtained by the following measurement conditions (1) and (2) using a differential scanning calorimeter (DSC) having an ultimate viscosity (η). It is desirable that the temperature at which the heat absorption peak is not observed or the heat absorption peak is confirmed exceeds -1.4 ° C and is 1.1 ° C or less.
(Here, DSC measurement conditions:
(1) After holding at 20 ° C for 1 minute, the temperature drops to -80 ° C at a temperature lowering rate of 5 ° C / min, and (2) after holding at -80 ° C for 1 minute, the temperature rises to 20 ° C at a heating rate of 10 ° C / min. Temperature rising. ). The normal projection area of the biological sample in the state of being solidified at -80 ° C in the polymer aqueous solution is 8 / compared with the normal projection area of the biological sample in the culture medium (in the medium) before lowering the temperature (before freezing). It is reduced to less than 10, preferably 1 / 3.5 or more.

Since the polymer aqueous solution of the present invention used in the cryopreservation method using the polymer aqueous solution of the present invention has a glass transition temperature in the vicinity of -23 ° C ± 4 ° C, its vitrification is frozen. Since it is sometimes realized by trapping water molecules in a network of water-soluble polymers, the cryopreservation method using the aqueous polymer solution of the present invention does not require a large cooling rate. Therefore, after the biological sample is contained in the polymer aqueous solution of the present invention, the polymer aqueous solution of the present invention is cooled to −27 ° C. or lower, which is the glass transition temperature, for example, the polymer aqueous solution of the present invention containing the biological sample. Is simply placed in a freezing treatment container and left in a deep freezer at -80 ° C, and the cells and tissues to be stored are stably frozen and stored as a frozen solidified body while maintaining the survival rate. can do.

However, the range of the cryopreservation temperature is not limited as long as it is −27 ° C. or lower. For example, the upper limit is preferably −70 ° C. or lower, preferably −80 ° C. or lower. The lower limit is preferably -196 ° C. or higher, preferably −150 ° C. or higher.

The cryopreservation method using the aqueous polymer solution of the present invention is a slow freezing method. That is, in the freezing method using the aqueous polymer solution of the present invention, the cooling rate is preferably 10 ° C./min or less. More preferably, the cooling rate is about 1 ° C./min or less. At this cooling rate, it is considered that the intracellular dehydration is moderately performed, the intracellular fluid is vitrified well, and a high cell freeze-protection effect can be obtained.

Further, the pH of the aqueous polymer solution of the present invention may be adjusted, if necessary, before contacting with the biological sample. For example, when the hydrophilic group of the monomer having a hydrophilic group is a carboxylic acid group or the like, the aqueous polymer solution containing the water-soluble polymer on which such a monomer is polymerized may be acidic. Further, when the saccharide has a carboxylic acid group or the like, the polymer aqueous solution may be acidic due to such a saccharide. In such a case, it is considered that by adjusting the pH to make the aqueous polymer solution a neutral solution, the solution can be more suitable for the survival of the cells to be cryopreserved, and the survival rate of the cells is improved. The salt used for pH adjustment is not limited to that, and those usually used for pH adjustment of an aqueous solution can be used.

In the method for cryopreserving a biological sample using the aqueous polymer solution of the present invention, the vitrified state is stabilized and the toxicity is low, so that the biological sample can be stably stored for a long period of time. In the present specification, long-term stable storage means, for example, that the survival rate of a biological sample after thawing when the polymer aqueous solution of the present invention is used, for example, the survival rate of cells is based on the survival rate of cells immediately before storage. After 5 months, a decrease of less than 10%, preferably less than 5%, or after 6 months, a decrease of less than 20%, preferably less than 10%, or after 12 months, less than 15%. It means that only a decrease of about 30% or less is observed. Further, in the present specification, long-term stable storage means, for example, when cells are frozen and stored for a long period of time at -80 ° C, then thawed, and then the cells are stored at 4 ° C, after thawing. It means that even after 24 hours, the survival rate is reduced by less than 5% based on the cell survival rate immediately after thawing. It is considered that the cryopreservation method using the polymer aqueous solution of the present invention can cryopreserve cells under less stress conditions as compared with the conventional cryopreservation method using a DMSO solution as a cryopreservation solution. Therefore, in the cryopreservation method of the present invention, a very high cell viability after thawing can be obtained as compared with the cryopreservation method using a conventional cryopreservation solution such as a 10% DMSO solution. Furthermore, cells stored refrigerated after thawing as well as immediately after thawing can show high cell viability. According to the cryopreservation method using the aqueous polymer solution of the present invention, cells can be cryopreserved stably for a long period of time without changing the properties of the cells.

The present invention will be specifically described based on examples, but the present invention is not limited thereto. Further, in the following, the hyaluronic acid manufactured by lifecore biomedical is sodium hyaluronate, but for the sake of simplicity, it is described as "hyaluronic acid".

Water-soluble polymer and aqueous polymer solution for cryopreservation <Preparation of test sample and sample solution>
-Example 1
High molecular weight hyaluronic acid (manufactured by Shanghai Easier Industrial Development) having an average molecular weight of 1 million and water are mixed in a pressure-resistant container having a volume of 2 L at a treatment temperature of 175 ° C., a treatment pressure of 0.89 MPa, and a treatment time of 3. Subcritical treatment was performed in minutes. Then, the subcritical treated product was dried by freeze-drying (this drying was also possible by the spray-drying method). As a result, a water-soluble polymer was obtained, which was a mixture of a high molecular weight hyaluronic acid having a viscosity average molecular weight of about 10,000 and a hyaluronic acid having a viscosity average molecular weight of 1000.

When this water-soluble polymer was confirmed by HPLC (FIG. 12), the test sample of Example 1 was found to be a mixture of a high molecular weight hyaluronic acid decomposition product and a low molecular weight hyaluronic acid decomposition product by subcritical treatment of hyaluronic acid. Therefore, high molecular weight hyaluronic acid was precipitated in ethanol, low molecular weight hyaluronic acid was fractionated to the supernatant side, and the fractionated supernatant was reanalyzed by HPLC. Since it was almost the same as the HPLC peaks of hyaluronic acid (FIGS. 11A to 11C) of Production Examples 1 to 3 described later, it was estimated that the viscosity average molecular weight of the low molecular weight hyaluronic acid component obtained in Example 1 was 1000. ..

The ultimate 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 was water) as a solvent. , The concentration of the hyaluronic acid sample in the test sample solution is 10 w / v%). Although αMEM medium is used as a solvent in Examples, Production Examples and Comparative Examples of the present invention including this Example 1, ultrapure water may be used as a solvent instead of αMEM medium. Further, in the measurement of DSC described later, the solvent is ultrapure water instead of αMEM. Each test sample obtained by the following example was prepared and used at a predetermined concentration by using an appropriate solvent in each of the tests for evaluation described later.

-Manufacturing example 1
The same operation as in Example 1 was carried out except that the subcritical treatment was carried out with a treatment time of 7 minutes to obtain a test sample of a hyaluronic acid fragment having a viscosity average molecular weight of 1000. The ultimate viscosity was 0.08 dL / g.

-Manufacturing example 2
The same operation as in Example 1 was carried out except that the subcritical treatment was carried out with a treatment time of 5 minutes to obtain a test sample of a hyaluronic acid fragment having a viscosity average molecular weight of 2000. The ultimate viscosity was 0.14 dL / g.

-Manufacturing example 3
The same operation as in Example 1 was carried out except that the subcritical treatment was carried out with a treatment time of 4 minutes to obtain a test sample of a hyaluronic acid fragment having a viscosity average molecular weight of 3000. The ultimate viscosity was 0.19 dL / g.

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

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

-Comparative example 3
Test sample of Comparative Example 3 by dissolving 1 g of gelatin (viscosity average molecular weight 315000 manufactured by Nitta Gelatin Co., Ltd.) in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, solvent is water). A solution was obtained (ie, the gelatin concentration of the test sample in the test sample solution was 10 w / v%). The ultimate viscosity was 0.50 dL / g.

-Comparative example 4
Comparative Example 4 by dissolving 1 g of high molecular weight hyaluronic acid (manufactured by Shanghai Easier Industrial Development) having a viscosity average molecular weight of 1000000 in 100 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, solvent is water) as a solvent. (I.e., the hyaluronic acid concentration of the test sample in the test sample solution was 1 w / v%). The ultimate viscosity was 17.2 dL / g.

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

-Comparative example 6
Chondroitin sodium sulfate A (manufactured by sigma) having a viscosity average molecular weight of 23000 was used as a test sample. By dissolving 1 g of this test sample in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent is water), the test sample solution of Comparative Example 6 was obtained (that is, the test sample). The chondroitin sulfate concentration of the test sample in the solution is 10 w / v%). The ultimate viscosity was 0.97 dL / g.

-Comparative example 7
A test sample of Production Example 1 (a hyaluronic acid fragment sample having a viscosity average molecular weight of 1000) was added to a carboxypolylysine (viscosity average molecular weight 13400: CryoScarless DMSO free manufactured by Bioverde Co., Ltd.) in which an amino group was 60% carboxylated. It was added in an amount of 1 w / v% to prepare a test sample solution of Comparative Example 7. The ultimate viscosity was 0.11 dL / g.

-Comparative example 8
Fructan derived from scallions was used as a test sample. Fructans were obtained by extraction and purification by the following method according to Example 2 of JP2012-235728.

About 1 kg of dried scallions as a raw material was crushed, mixed with water for extraction in which 15 g of calcium hydroxide was suspended, extracted at room temperature for 1 hour, and then heat-extracted at 95 ° C. for 1 hour. Then, it was extracted by heating at 60 ° C. overnight. Activated carbon was added to the extract for deodorization treatment, and then filtration was performed with diatomaceous earth to remove the residue and activated carbon. The obtained extract was passed through an ultrafiltration membrane to purify fructan.

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

The viscosity average molecular weight of the obtained purified fructan powder was measured and found to be 12000.

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

-Comparative example 9
Pullulan (manufactured by Tokyo Chemical Industry Co., Ltd.) having a viscosity average molecular weight of 373000 was used as a test sample. By dissolving 1 g of this test sample in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent is water), the test sample solution of Comparative Example 9 was obtained (that is, the test sample solution). The hyaluronic acid concentration of the test sample in the sample is 10 w / v%). The ultimate viscosity was 0.55 dL / g.

-Example 2
Hyaluronic acid concentration 10 w / v% in which 1 g of a test sample (hyaluronic acid having a viscosity average molecular weight of 15,000) of Comparative Example 5 was dissolved in 10 mL of an αMEM medium (manufactured by Gibco, product number C1257-1500BT, solvent is water). The test sample of Production Example 1 (hyaluronic acid fragment sample having a viscosity average molecular weight of 1000) was added to the test sample solution of No. 1 at a final concentration of 1 w / v%. The pH of the obtained aqueous solution was adjusted to neutral with 10 mM Tris-HCl to prepare a test sample solution of Example 2. The ultimate viscosity was 0.65 dL / g.

-Example 3
A test with a pull run concentration of 10 w / v% in which 1 g of a test sample (pull run having a viscosity average molecular weight of 373000) of Comparative Example 9 was dissolved in 10 mL of an αMEM medium (manufactured by Gibco, product number C1257-1500BT, solvent is water). A test sample of Production Example 1 (a hyaluronic acid fragment sample having a viscosity average molecular weight of 1000) was added to the sample solution for test at a final concentration of 1 w / v% to obtain a test sample solution of Example 3. The ultimate viscosity was 0.55 dL / g.

-Example 4
A test with a pull run concentration of 10 w / v% in which 1 g of a test sample (pull run having a viscosity average molecular weight of 373000) of Comparative Example 9 was dissolved in 10 mL of an αMEM medium (manufactured by Gibco, product number C1257-1500BT, solvent is water). A test sample (a hyaluronic acid fragment sample having a viscosity average molecular weight of 1000) of Production Example 1 was added to the sample solution at a final concentration of 5 w / v%. The pH of the obtained aqueous solution was adjusted to neutral with 10 mM Tris-HCl to prepare a test sample solution of Example 4. The ultimate viscosity was 0.55 dL / g.

Example 5
High molecular weight hyaluronic acid (manufactured by Shanghai Easier Industrial Development) having an average molecular weight of 1 million and water are mixed in a pressure-resistant container having a volume of 2 L at a treatment temperature of 175 ° C., a treatment pressure of 0.89 MPa, and a treatment time of 3. Subcritical treatment was performed in 5. minutes. Then, the subcritical treated product was dried by freeze-drying or spray-drying method. As a result, a hyaluronic acid sample having a viscosity average molecular weight of about 6000 was obtained.
Sculose (Nakaraitesku Co., Ltd.) was added to a test sample solution having a hyaluronic acid concentration of 10 w / v% in which 1 g of this hyaluronic acid was dissolved in 10 mL of an αMEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent was water). ), Molecular weight (same value as viscosity average molecular weight and imitation) is 342), and glucuronic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., molecular weight (viscosity average molecular weight and imitation) 194) are each with a final concentration of 1 w / v%. The mixture was added in an amount to obtain a test cryopreservation solution of Example 5. The ultimate viscosity was 0.32 dL / g.

-Example 6
Hyaluronic acid concentration 10 w / v% in which 1 g of a test sample (hyaluronic acid having a viscosity average molecular weight of 15,000) of Comparative Example 5 was dissolved in 10 mL of an αMEM medium (manufactured by Gibco, product number C1257-1500BT, solvent is water). A test sample of Production Example 2 (a hyaluronic acid fragment sample having a viscosity average molecular weight of 2000) was added to the aqueous solution of Example 2 at a final concentration of 1 w / v% to obtain a test sample solution of Example 6. The ultimate viscosity was 0.47 dL / g.

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

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

FIG. 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. It shows the cell viability after thawing of the cells that have been thawed. As shown in FIG. 1, in Example 1, that is, the viscosity average molecular weight obtained by the subcritical treatment of hyaluronic acid is about 10,000, and the viscosity average molecular weight is 1000, that is, smaller than 10,000. The hyaluronic acid sample containing the cleavage product of hyaluronic acid having a molecular weight is a water-soluble polymer in a high molecular weight aqueous solution, has an ultimate viscosity of 0.49 dL / g, and generates heat in DSC measurement during the temperature lowering process. In the test sample solution of Example 1 having an amount of 0 J / g (see Test Example 2 below), a remarkably high storage effect of more than 90% was obtained.

In the frozen solidified body obtained by freezing the test sample of Example 1, no endothermic peak was confirmed in the DSC measurement during the heating process (see Test Example 2 below), so that the water molecules formed ice crystals. It is presumed that the water is bound by being adsorbed or entangled in free water or hyaluronic acid molecular chains.

In addition, since the difference in brightness between the solvent matrix region and the cell region of this frozen solidified product is as small as 2 (see Test Example 8 below), the existence state of water molecules in the solvent matrix region and the cell region is approximated, and the intracellular state is approximated. It is considered that ice crystals are not formed. Furthermore, when the test sample solution is solidified at -80 ° C, the normal projection area of the cells is shrunk to 38% of the normal projection area of the cells in the culture medium (in the medium) before solidification (see below). From (see Test Example 9), it is considered that hyaluronic acid has not invaded the cell and water molecules have moved to the outside of the cell due to the osmotic pressure difference. Water molecules that have moved to the outside of the cell are thought to replace low-molecular-weight hyaluronic acid or high-molecular-weight hyaluronic acid, and remain around the cell to form ice crystals, which may cause problems such as destruction of cell tissue. Also, hyaluronic acid does not enter the cell and harm the cell. Therefore, unlike DMSO of Comparative Example 1, the cytotoxicity is low and ice crystals are not formed in the cell and around the cell, so that the cryopreservation effect of the cell is also excellent.

The preservation effect of this test sample solution was even higher than that of Comparative Example 1 containing 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 having low cytotoxicity.

As can be understood from Table 1, from the comparison between Examples 3 and 4 and Comparative Example 9, the calorific value measured by DSC in the temperature lowering process of the aqueous polymer solution is 65 of that of standard water (ultrapure water). If it exceeds%, or if the amount of heat absorbed by DSC measurement in the process of raising the temperature of the frozen solidified body exceeds 65% of that of standard water (ultrapure water), conditions such as extreme viscosity and cell shrinkage rate are required. Even if it is satisfied, the viability of the cells will decrease.

The test sample solution of Comparative Example 4 contained high molecular weight hyaluronic acid having a large molecular weight of 1,000,000 as a test sample, but the ultimate viscosity was as large as 17.2 dL / g, and an endothermic peak was not confirmed (the following test example). Despite this), almost no cytoprotective effect was observed.

In the frozen solidified product of Comparative Example 4, no endothermic peak was confirmed in the DSC measurement during the heating process, but no shrinkage was observed in the frozen solidified cells, and therefore water molecules remained in the cells. it is conceivable that. In addition, the difference in brightness between the solvent matrix region and the cell region of the frozen solidified product is as large as 4 (see Test Example 8 below), the cell region is darkened, and water molecules in the cell form ice crystals. it is conceivable that. Therefore, it is presumed that the ice crystals destroyed the cells and the preservation effect of the cells was not observed.

In Comparative Example 2 containing no cryopreservative, the cell viability was almost 0%.

Further, in Comparative Example 7 containing carboxypolylysine as a test sample and having a small ultimate viscosity of 0.11 dL / g, the cytoprotective effect as in Example 1 and Comparative Example 1 using DMSO as a cryoprotectant was not observed. In the test sample solution of Comparative Example 7, since the extreme viscosity of polylysine is low, water molecules that have moved to the outside of the cell and pericellular high molecular weight or low molecular weight saccharides are diffused, and the water molecule and the high molecular weight or low molecular weight are diffused. It is presumed that the replacement with saccharides having a molecular weight does not proceed, and water molecules remain around the cells to form ice crystals. In addition, since the area of the cells after freezing is larger than the area in the culture medium (in the medium) (see Test Example 9 below), polylysine has invaded the cells or the cells are formed by ice crystals. It is thought that the cells are expanding. For this reason, it is speculated that the cells may be damaged and a sufficient preservation effect may not be obtained.

Further, in Comparative Example 8 using fructan, the ultimate viscosity was as small as 0.11 dL / g, and the replacement of water molecules that had moved to the outside of the cell with the pericellular polymer or low molecular weight saccharide did not proceed. It is thought that about half of the cells burst and die due to ice crystal formation.

From Example 5, it is understood that glucuronic acid and sucrose also have a function of adjusting the endothermic peak to around 0.7 ° C. (The endothermic peak of hyaluronic acid having a viscosity average molecular weight of about 6000 is -1. 4 ° C).

Further, since the polymer aqueous solution of the present invention showed a high cell preservation effect even when prepared using a culture solution such as αMEM medium instead of water as a solvent, the polymer aqueous solution of the present invention was used as it was for cells. After suspending the cells in addition to the culture medium of, the cells can be frozen and stored. It is considered possible to maintain higher cell viability and cell properties without the need to centrifuge the cells to be preserved.

<Test Example 2: Analysis of each test sample solution by a differential scanning calorimeter>
The solvent of each test sample solution of Examples 1 to 6, Comparative Examples 1 to 9 and Production Examples 1 to 3 was changed from αMEM medium to ultrapure water to prepare a sample for differential scanning calorimetry. Each sample was scanned with a differential scanning calorimetry device (DSC) as shown below.
(1) After holding at 20 ° C for 1 minute, the temperature is lowered to -80 ° C at free speed.
(2) After holding at -80 ° C for 1 minute, the temperature is raised to 20 ° C at a heating rate of 10 ° C / min.

In the case of Example 1, Comparative Example 5 and ultrapure water (Comparative Example 2), the DSC curve of the temperature rising process obtained by the above DSC measurement conditions is shown in FIG. 2A. FIG. 2B is an enlarged view of FIG. 2A in the vicinity of the glass transition temperature of the aqueous polymer solution of the present invention. Further, in the case of Example 1, Comparative Example 1 and Ultrapure Water (Comparative Example 2), the DSC curves of each are shown in FIGS. 3A to 3C.

Further, in the DSC curve of the temperature rise process of each test sample solution, the temperature at which the endothermic peak estimated to be caused by the melting of water is observed, the endothermic amount of the endothermic peak (heat absorption amount at the time of temperature rise), and the temperature lowering process. The calorific value of the exothermic peak observed in the DSC curve (calorific value at the time of temperature decrease) was analyzed. The results are shown in Table 1. In Table 1, the endothermic amount of the endothermic peak is shown by the ratio (%) when the endothermic amount of 112.4 J / g measured with ultrapure water (Comparative Example 2; used as a reference liquid) is set to 100. .. Further, in Table 1, the calorific value of the exothermic peak is shown by the ratio (%) when the calorific value 106 J / g measured with ultrapure water (Comparative Example 2; used as a reference liquid) is set to 100.

As shown in FIGS. 2A and 2B, in Comparative Example 2 in which the test sample was ultrapure water (that is, the sample in which the test sample was ultrapure water), only the ice crystal melting peak of free water was observed (FIG. 2A). ). In Comparative Example 5 in which the test sample was hyaluronic acid having a viscosity average molecular weight of 15,000, a shift in the ice crystal melting peak and a freezing point depression were observed as compared with Comparative Example 2 (FIG. 2A), and glass was observed at around -20 ° C. Transfer was confirmed (Fig. 2B). On the other hand, in the test sample of Example 1 containing a cleavage product of hyaluronic acid (low molecular weight hyaluronic acid having a viscosity average molecular weight of 1000) in addition to hyaluronic acid having a viscosity average molecular weight of about 10,000, the peak of ice crystal melting was It was extremely small and almost non-existent (Fig. 2A), and only a glass transition was confirmed around -23 ° C ± 4 ° C (Fig. 2B). This result shows that in the aqueous polymer solution of Example 1, water exists in the matrix without freezing (free water) or in a state of being entangled with the polymer (bound water). It shows a remarkably advantageous effect of the aqueous polymer solution of the present invention that the recrystallization of water at the time of melting is suppressed.

Further, as shown in FIG. 3A, in Comparative Example 2 in which the test sample was ultrapure water, a very large exothermic peak associated with ice crystal formation was observed in the temperature lowering process. Also, in the process of raising the temperature, a large amount of heat of melting 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 peaks associated with small ice crystal formation were observed, and few peaks associated with ice crystal melting were observed. This indicates that the cryopreservation solution containing the test sample (DMSO) of Comparative Example 1 is close to the vitrified state in the frozen state. In the high molecular weight aqueous solution of the test sample of Example 1 (hyaluronic acid having a viscosity average molecular weight of 10000 containing a cleavage product of hyaluronic acid), as shown in FIG. 3C, almost no melting peak was observed, and ice crystals were observed. The peaks associated with the formation were also much smaller than the peaks observed in Comparative Example 1. Therefore, it is considered that ice crystals are hardly formed in the polymer aqueous solution containing the test sample of Example 1, and the vitrification state is extremely good.

The temperature at which the heat absorption peak was observed (heat absorption peak temperature) was −1.4 ° C. in Comparative Example 5 (the test sample was hyaluronic acid having a viscosity average molecular weight of 15,000). In Example 2 in which a hyaluronic acid fragment having a viscosity average molecular weight of 1000 or less was mixed, the temperature was −0.4 ° C., which was close to the temperature at which the heat absorption peak of pure water appeared at 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 having an endothermic peak appearance temperature lower than that of pure water, the endothermic peak appearance temperature is expected to be further lowered, but the opposite is true. In addition, the endothermic peak appearance temperature can be adjusted so that it approaches the endothermic peak appearance temperature of pure water or does not appear the endothermic peak or the exothermic peak itself.

On the other hand, the endothermic peak appearance temperature of Comparative Example 9 (the test sample was pullulan having a viscosity average molecular weight of 373000) was 1.49 ° C, which was higher than the endothermic peak appearance temperature of pure water of 0.7 ° C. When Production Example 1 (endothermic peak appearance temperature is −8.1 ° C.) is mixed with this Comparative Example 9 (Examples 3 and 4), the endothermic peak appearance temperature is lowered instead of increased, and the pure water is used. The endothermic peak appearance temperature was 0.83 ° C, which is close to 0.7 ° C.

That is, when hyaluronic acid having a molecular weight of 1000 or less and a water-soluble polymer having a heat absorption peak appearance temperature lower than that of pure water are mixed, the peak appearance temperature of the aqueous polymer solution rises and is brought closer to pure water, and the hyaluronic acid having a molecular weight of about 1000 is used. It was clarified that when a water-soluble polymer having a heat absorption peak appearance temperature higher than that of pure water is mixed, the peak appearance temperature of the aqueous polymer solution is lowered to bring it closer to 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 Example 6 in which the low molecular weight sample of Production Example 2 was mixed with Comparative Example 5 (endothermic peak appearance temperature was −1.4 ° C.) was lowered to −1.6 ° C.

This result shows that the heat absorption peak temperature of the polymer aqueous solution is adjusted by using a low molecular weight saccharide or a salt thereof, particularly preferably a low molecular weight saccharide having a viscosity average molecular weight of 3000 or less. It is shown that the heat absorption peak temperature of pure water can be set to around 0.7 ° C. When sucrose, trehalose, or glucuronic acid was used as a low-molecular-weight saccharide having a viscosity average molecular weight of 3000 or less or a salt thereof, the same effect as that of the sample of Production Example 1 was observed with respect to the heat absorption peak temperature.

Further, the calorific value of the exothermic peak in the temperature lowering process of the test sample solutions of Examples 1 to 6 was smaller than the corresponding calorific value of the reference liquid consisting of water (Comparative Example 2), and was 65% or less. Further, the endothermic amount of the endothermic peak in the process of raising the temperature of the test sample solution of Examples 1 to 6 was smaller than the corresponding endothermic amount of the reference liquid composed 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 solidified in a glass state without being crystallized.

The endothermic peak temperature of the test sample solution of Examples 1 to 6 is near the endothermic peak temperature of water of 0.7 ° C., that is, the behavior is close to that of pure water, and the calorific value of the exothermic peak is water. The fact that it is smaller than the calorific value of the exothermic peak observed in the temperature lowering process indicates that bound water or free water that does not form ice crystals remains in the frozen polymer aqueous solution of the present invention. It is considered that the water-soluble polymer in the present invention realizes vitrification by trapping water molecules in the matrix when frozen.

<Test Example 3: Evaluation of long-term storage effect in cryopreservation using a test sample solution of primary human mesenchymal stem cells>
The 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.} Then, the cell suspension containing each test sample solution was frozen in a slow cell freezer (Nalgene (registered trademark) Mr. Frosty) in a -80 ° C freezer to obtain a frozen solidified product. After storing the frozen cell suspension containing each test sample solution at -80 ° C for up to 12 months, the cell suspension containing each test sample solution is taken out at a predetermined storage period at 37 ° C. Rapidly thawed in a warm bath. The cell viability of the cell suspension containing each test sample solution after thawing was evaluated by trypan blue staining immediately after thawing. The results are shown in FIG. 4A. In addition, the cell suspension cryopreserved at −80 ° C. for 3 months was thawed, and then stored at 4 ° C. for 1 day or 1 week. The cell viability of the cell suspension after storage at 4 ° C. was evaluated by trypan blue staining. The cell viability after storage at 4 ° C. was calculated assuming that the cell viability immediately after thawing (that is, immediately before storage) was 100%. The results are shown in FIG. 4B.

From the results shown in FIG. 4A, in the cryopreservation using the aqueous polymer solution of the present invention, the frozen cells showed a high cell viability of 95% or more, which was almost unchanged even after storage for 5 months. 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 the cell viability was already observed 2 months after the cryopreservation. Then, after 3 months, it can be seen that the cell viability in the cryopreservation of Comparative Example 1 was less than 50%. This result was in contrast to the aqueous polymer solution of the present invention, which still maintained a high cell viability even after 3 months. After 6 months of cryopreservation, cryopreservation using the polymer aqueous solution of the present invention showed a decrease in survival rate of less than 10%, whereas cryopreservation using the sample solution of Comparative Example 1 showed cells. The survival rate had dropped to about 25%. Furthermore, in the cryopreservation in the aqueous polymer solution of the present invention, the high cell viability was still maintained even after 12 months of the cryopreservation, and the decrease in the cell viability was 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 viability.

Further, when cells stored at -80 ° C for a long period of time are thawed and then stored at 4 ° C as they are, when the polymer aqueous solution of the present invention is used, the cell viability after storage at 4 ° C for 1 day is immediately before storage. While it was close to 100% of the cell viability in Comparative Example 1, it decreased to about 60% in Comparative Example 1. In the cells cryopreserved using the aqueous polymer solution of the present invention, cell viability of 40% or more was still observed even after storage at 4 ° C. for one week. This is because the cells that survived after thawing in the cryopreservation using the polymer aqueous solution of the present invention were cells that proceeded to proliferate substantially normally, and the polymer aqueous solution of the present invention became cells during storage. It was shown that there was no damage to the cells, which confirmed that the polymer aqueous solution of the present invention had a very excellent cell preservation effect.

<Test Example 4: Evaluation of properties of primary human mesenchymal stem cells after cryopreservation using a test sample solution>
The 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.} Then, the cell suspension containing each test sample solution was frozen in a slow cell freezer (Nalgene (registered trademark) Mr. Frosty) in a -80 ° C freezer to obtain a frozen solidified product. Cell suspensions containing frozen test sample solutions were cryopreserved at −80 ° C. for 6 months. After 6 months, the cell suspension containing each test sample solution was rapidly thawed in a warm bath at 37 ° C., washed with αMEM, and then 2.5 × 10 ⁴ cells were seeded in 6-well plates 4 days later. , HGF and IL-10 concentrations in culture medium were quantified. For quantification of HGF and IL-10, dedicated kits (Quantikine® ELISA Human HGF, catalog number DHG00, manufactured by R & D), Quantikine® ELISA Human IL-10, catalog number D100B, R & D, respectively. The procedure was performed according to the order of the procedure manual attached to the kit. The results are shown in FIGS. 5A and 5B.

As shown in FIG. 5A, in the cryopreservation in the presence of the sample solution of Comparative Example 1 containing DMSO as a cryopreservative, HGF production in the cells after thawing was high. On the other hand, in the cryopreservation using the test sample solution of Example 1 of the present invention, which is a hyaluronic acid having a viscosity average molecular weight of 10,000 containing a cleavage product of hyaluronic acid, the amount of HGF produced in the cells after thawing was low, and the comparative example. It was less than 1/3 of 1. The results in the presence of DMSO indicate that the cells were under stress when stored in the presence of DMSO. Since such high HGF production is not observed in the cell storage using the polymer aqueous solution of the present invention, it can be seen that the cells are stably protected by the polymer aqueous 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 cells cryopreserved using the test sample solution of Comparative Example 1. Then it was very low. From this result, it can be seen that the aqueous polymer solution of the present invention can satisfactorily cryopreserve cells while maintaining the functions of the cells.

Since the aqueous polymer solution of the present invention does not contain compounds having cytotoxicity such as DMSO and ethylene glycol, the aqueous polymer solution of the present invention is different from the conventional cryopreservation solution without exposing the biological sample to a stress state. It has a remarkable effect that it can be cryopreserved while maintaining its properties.

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

<Test Example 5: Evaluation of properties (undifferentiated) after cryopreservation using a test sample solution of primary human mesenchymal stem cells>
The 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.} Then, the cell suspension containing each test sample solution was frozen in a slow cell freezer (Nalgene (registered trademark) Mr. Frosty) in a -80 ° C freezer to obtain a frozen solidified product. Cell suspensions containing frozen test sample solutions were cryopreserved at −80 ° C. for 6 months. After 6 months, the cell suspension containing each test sample solution was removed and rapidly thawed in a warm bath at 37 ° C. The expression intensities of CD90, CD44 and CD105 were analyzed by flow cytometry in the cell suspension containing each test sample solution after thawing. The results are shown in FIG.

CD90, CD44 and CD105 are typical surface proteins expressed in undifferentiated mesenchymal stem cells and are used as undifferentiated markers of mesenchymal stem cells. As shown in FIG. 6, the expression of all undifferentiated biomarkers of CD90, CD44 and CD105 was cryopreserved in the test sample solution of Comparative Example 1 in the cells cryopreserved in the polymer aqueous solution of Example 1. It was higher than the cells that were used. Therefore, it can be seen that the cells cryopreserved in the polymer aqueous solution of Example 1 maintain the expression of the undifferentiated marker. That is, the cells cryopreserved in the polymer aqueous solution of Example 1 can maintain an undifferentiated state. It can be seen that the cryopreservation using the polymer aqueous solution of the present invention can reduce the influence on the differentiation state as seen when stored in the test aqueous solution of Comparative Example 1.

From this result, it can be seen that the cryopreservation method using the aqueous polymer solution of the present invention is suitably applicable to the cryopreservation of stem cells in which it is important to cryopreserve 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) at ^{a concentration of 1 × 10 6} cells / mL, test sample solutions of Examples 1 to 4 and 6 and Comparative Examples 3, 6 and 9 (serum-free). (However, the concentration of the hyaluronic acid sample in the test sample solution of Example 1 was set to 20 w / v%). Then, the cell suspension containing each test sample solution was frozen in a slow cell freezer (Nalgene (registered trademark) Mr. Frosty) in a -80 ° C freezer to obtain a frozen solidified product. After cryopreservation for 1 day, the cell suspension containing each test sample solution was taken out and rapidly thawed in a warm bath at 37 ° C. 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 results of the cell viability shown in Table 1 together with the results of Test Example 1, the water-soluble polymer in the present invention is not limited to hyaluronic acid, and even other water-soluble polymers have high concentrations. It can be seen that if the aqueous molecular solution has a predetermined extreme viscosity and heat absorption peak temperature, it exhibits a high cytoprotective effect. Further, from the results of Comparative Example 9 and Examples 3 and 4 (Comparative Example 9 to which Production Example 1 was added), a water-soluble polymer was used in combination with a saccharide having a smaller molecular weight or a salt thereof. , It was found that the polymer aqueous solution shows a good cytoprotective effect. As shown in the results of Test Example 2, this is because the endothermic peak temperature of the sample solution is changed to the endothermic peak temperature of pure water by 0.7 ° C. by adding a low molecular weight saccharide or a salt thereof to the aqueous polymer solution. It is presumed that it was possible to adjust it so that it was in the vicinity. Example 6 is a test sample obtained by adding a low molecular weight saccharide (a hyaluronic acid fragment sample having a viscosity average molecular weight of 2000) of Production Example 2 to a high molecular weight aqueous solution of hyaluronic acid having a viscosity average molecular weight of 15,000 (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, which is lower than the endothermic peak temperature (-1.4 ° C) of Comparative Example 5. As a result, the endothermic peak appearance temperature of pure water has moved away from 0.7 ° C. Also, in this test example 6, the cell viability as high as that of the other examples was not obtained in the example 6. From this result, it is important to mix a low molecular weight saccharide or a salt thereof with a high molecular weight aqueous solution to adjust the endothermic peak temperature of the test sample solution to a desired range, whereby a high cell viability can be obtained. I found out that I would get it.

In addition, when the test sample solution prepared by adding a saccharide or a salt thereof is acidic, neutralizing the test sample solution by adjusting the pH has a good cytoprotective effect. Obtained. It is considered that the pH adjustment made the conditions more suitable for cell survival. In order for the added low molecular weight saccharide or salt thereof to act as a cytoprotective component and further improve the cell viability of the cells after cryopreservation, the low molecular weight saccharide or salt thereof is added to the aqueous polymer solution. It was necessary to add it in an amount of 1 to 5 w / v% with respect to the water-soluble polymer. On the other hand, even if the amount of the low molecular weight saccharide or the salt thereof to be added was larger than 10 w / v% with respect to the water-soluble polymer, no further effect was observed on the effect of improving the cell viability.

From Table 1, the ultimate 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. Below, it can be seen that when the temperature is higher than 1.1 ° C., the cell viability after cryopreservation decreases. As is clear from Table 1, even if only one of the extreme viscosity and the endothermic peak temperature is within a predetermined range, the desired effect (high cell viability) cannot be obtained. When the ultimate viscosity (η) is within the above-mentioned predetermined range, the aqueous polymer solution assists the discharge of water molecules from the biological sample, and efficiently dissipates the discharged water molecules to the height around the biological sample. It is believed that it can be replaced with molecular and / or small saccharides. Furthermore, the fact that the endothermic peak temperature exceeds -1.4 ° C and is about 1.1 ° C or less is considered to indicate that the aqueous polymer solution can retain water molecules in the polymer matrix during freezing. Be done.

In addition, from the results of Test Example 6, it was found that the aqueous polymer solution of the present invention exhibits a good cryoprotective effect not only on humans but also on canine mesenchymal stem cells.

<Test Example 7: Evaluation of cells in frozen test sample solution (evaluation of intracellular vitrification state)>
The 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. Then, 5 μL of the cell suspension and the control suspension containing each test sample solution were added to the hard glass sample plate (16φ × 0.12 mm), and the hard glass cover glass (12φ × 0.12 mm) was added. The temperature was lowered to −80 ° C. at 5 ° C./min on a cooling stage for a microscope (THMS600) manufactured by LinKam, and an image was taken at −80 ° C. with a transmitted light microscope (Olympus BX53). The results are shown in FIGS. 7A-C.

FIG. 7A shows the results of the control suspension, and it can be seen that the cells are darkened due to the generation of ice crystals in the cells and the diffuse reflection of light in the medium alone. FIG. 7B shows the results of Comparative Example 1 in which DMSO is a test sample. Also in FIG. 7B, it can be seen that the cells are darkened and micro ice crystals are formed. FIG. 7C is a result of a polymer aqueous solution containing the test sample of Example 1. It can be seen that the cells are turning bright and the inside of the cells is vitrified into an amorphous state.

From this result, it can be seen that the aqueous polymer solution of the present invention vitrifies the inside of the cell and freezes it. It was found that the glass state was formed more stably by containing the saccharide which is a cleavage product of hyaluronic acid. It is considered that the formation of ice crystals around the cells was suppressed by the saccharides having a low molecular weight, so that vitrification occurred stably and efficiently.

<Test Example 8: Evaluation of intracellular vitrification state using difference in brightness>
The cultured primary canine mesenchymal stem cells (cyagen C160) were suspended in the test sample solutions (serum-free) of Examples 1 to 6 and Comparative Examples 1 to 9 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. Then, 5 μL of the cell suspension and the control suspension containing each test sample solution were added to the hard glass sample plate (16φ × 0.12 mm), and the hard glass cover glass (12φ × 0.12 mm) was added. The temperature was lowered to −80 ° C. at 5 ° C./min on a cooling stage for a microscope (THMS600) manufactured by LinKam, and an image was taken at −80 ° C. with a transmitted light microscope (Olympus BX53). 8A and 8B show the results of Example 1 and Comparative Examples 1 and 2.

The difference (absolute value) between the intracellular and extracellular light and darkness of the observation image shown in FIG. 8A was analyzed using the image analysis software ImageJ. In each image, the lightness of the solvent region and the lightness inside the cell and the Munsell lightness are read under the same conditions, and the read data of the lightness of the solvent region and the lightness inside the cell are compared with the read data of each Munsell lightness. Then, the Munsell lightness of the closest read data was adopted as the lightness of the solvent region and the lightness inside the cell, and the lightness of the solvent and the lightness inside the cell were quantified (Munsell value conversion). If the read value of the lightness of the intracellular region or the solvent region read by the image analysis software is darker than the read value of the darkest value 0 of the Munsell lightness, the Munsell of the intracellular region or the solvent region is conveniently read. When the value is set to 0 and the read value of the lightness of the intracellular region or the solvent region read by the image analysis software is a brighter value than the read value of the brightest value 10 of the Munsell lightness, the intracellular region or the solvent is used 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 between the Munsell brightness in the solvent region and the Munsell brightness in the cell internal 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 difference in Munsell brightness was 4. On the other hand, in Example 1, the Munsell brightness in the solvent region was 4, the Munsell value in the intracellular region was 2, and the difference in Munsell brightness was 2 (FIG. 8B). Similarly, for Examples 2 to 6 and Comparative Examples 3 to 9, the Munsell brightness in the solvent region and the Munsell brightness in the intracellular region were obtained, and the difference in brightness was calculated. The results are shown in Table 1.

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

Further, the difference in brightness between the Munsell brightness in the solvent region and the Munsell value in the intracellular region was 2 in Examples 2 to 6 as in Example 1. That is, in the examples, the brightness of the intracellular region and the brightness of the solvent region are close to each other. The result is that in the cryopreservation using the polymer aqueous solution containing the test sample of the example, the frozen state is almost the same in the matrix region and the intracellular region, that is, ice crystals are formed in the region in 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. Further, it is desirable that the lightness value of the solvent region is larger or the same as the lightness value of the intracellular region.

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

Cells frozen in the test sample solutions of Examples 1-6 showed cell contraction rates of 38%, 33%, 67%, 76%, 40% and 50%, respectively. In the cryopreservation using the test sample solution of Examples 1 to 6, the drainage of water from the cells to be frozen is promoted, and the cells are well dehydrated at the time of freezing. In the freezing of the biological sample with the aqueous polymer solution of Examples 1 to 6, in each case, the water-soluble polymer and / or the low molecular weight saccharide in the present invention is present sufficiently close to the periphery of the cell, and the intracellular fluid is present. Water molecules that have moved from the inside of the cell to the outside of the cell due to the osmotic pressure difference generated between the intracellular and the aqueous solution of the polymer are trapped in the matrix of the water-soluble polymer to further promote the discharge of water from the inside of the cell, and then inside the cell. The increased concentration of proteins and sugars in the cell overcools the intracellular water, and the difference in vapor pressure between the overcooled water further promotes the movement of water molecules through the cell membrane to the outside of the cell. Prevents or suppresses freezing and growth of ice crystals inside. Further, since the cells are contracted, it can be seen that the water-soluble polymer and the like do not invade the cells.

On the other hand, in Comparative Example 1 in which DMSO is a test sample and Comparative Example 2 in which DMSO is only a medium, the normal projection area of cells increased after freezing. Further, in the cryopreservation in the test sample solutions of Comparative Examples 3, 4 and 6 in which the ultimate viscosity (η) was outside the range of 0.20 dL / g or more and 0.95 dL / g or less, the cells were the same as before freezing. It had almost the same normal projection area. Further, even if the intrinsic viscosity (η) is in 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 of 0.7 ° C., and- Sufficient cell contraction was not observed in the test sample solutions (Comparative Examples 5 and 9) at 1.4 ° C or lower and higher than 1.1 ° C. The cryopreservation of these comparative examples in the test sample solution did not cause or was sufficient for cell dehydration, and therefore, the survival rate of the cells after cryopreservation was also good in Example 1 in which the cells were dehydrated. It is probable that it was lower than ~ 6. In Comparative Examples 7 and 8, the normal projection area of the cells was larger than that 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 considered that the water-soluble polymer has penetrated into the cells. In addition, the cell viability of the cells after cryopreservation in Comparative Examples 7 and 8 was also low. Such cell-permeable water-soluble polymers expand cells by freezing and damage cells, and are therefore suitable as water-soluble polymers for aqueous polymer solutions used for cryopreservation of biological samples. I can think that it is not. Further, in Production Examples 1 to 3 having a small ultimate viscosity (η), no change was observed in the normal projection area of the cells, and it was presumed that dehydration of the cells did not occur.

In the cryopreservation using the aqueous polymer solution of the present invention, the intracellular cells are well dehydrated at the time of freezing, so that the damage to the cells at the time of freezing is significantly reduced, whereby the thawed cells have a high survival rate. It is thought to show.

<Test Example 10: Evaluation of freeze protection for various cells>
Examples of cultured mouse-derived macrophage-like cell lines (RAW264), human colon cancer-derived cells (Caco-2), and primary human lung microvascular endothelial cells (HMVEC) at ^{a concentration of 1 × 10 6} cells / mL. It was suspended in the test sample solution (without serum) of 1 and Comparative Example 1. Then, the cell suspension containing each test sample solution was frozen in a slow cell freezer (Nalgene (registered trademark) Mr. Frosty) in a -80 ° C freezer to obtain a frozen solidified product. After cryopreservation for 7 days, the cell suspension containing each test sample solution was taken out and rapidly thawed in a warm bath at 37 ° C. 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 cryopreserved using the aqueous polymer solution of the present invention, the cell viability of all cells is as high as or higher than that of Comparative Example 1 in which DMSO is a test sample. Obtained. From this result, it was confirmed that the aqueous polymer solution of the present invention can cryopreserve various types of cells with high cell viability regardless of the primary cells or established cells, and regardless of the cell origin. Was done.

<Test Example 11: Evaluation of the effect of low molecular weight saccharides on cell viability>
The 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.} Then, the cell suspension containing each test sample solution was frozen in a slow cell freezer (Nalgene (registered trademark) Mr. Frosty) in a -80 ° C freezer to obtain a frozen solidified product. After cryopreservation for 7 days, the cell suspension containing each test sample solution was taken out and rapidly thawed in a warm bath at 37 ° C. 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 extreme viscosities (η) of low molecular weight saccharides or salts thereof (viscosity average molecular weights of 1000, 2000 and 3000, respectively) having a smaller molecular weight than the water-soluble polymer in the present invention are respectively. , 0.08, 0.14, 0.19, which is outside the desired range of 0.20 dL / g or more and 0.95 dL / g or less. From this Test Example 11, it was found that such a low molecular weight saccharide or a salt thereof alone cannot obtain a cell survival effect. In Production Example 3, a cell viability of about 10% was observed, which is presumed to be due to hyaluronic acid having a molecular weight higher than 3000 that may be contained in the test sample of Production Example 3.

<Test Example 12: HPLC analysis of subcritical 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 then the components of each test sample were analyzed by HPLC. As mobile phase, A solution: 16 mM NaH ₂ PO ₄ aqueous solution, B solution: 800 mM NaH ₂ with PO ₄ aqueous solution, ZORBAX NH ₂ (Agilent Technologies Co., column size φ4.6 × 250mm, particle size 5 [mu] m) The components were separated using a normal phase HPLC column at a flow velocity of 1.0 mL / min, a column temperature of 40 ° C., and a detection wavelength of 210 nm. The gradient condition was mobile phase B concentration 0% (0 minutes) → mobile phase B concentration 100% (60 minutes). The results are shown in FIGS. 11A-C. In addition, as a standard, HA02, which is a disaccharide, HA04, which is a tetrasaccharide, HA06, which is a hexasaccharide, HA08, which is an octasaccharide, and HA10, which is a ten sugar, at a concentration of 0.2 wt% (all manufactured by Izlon). Aqueous solutions containing hyaluronic acid oligosaccharides were prepared and subjected to HPLC analysis in the same manner. This result is shown in FIG. 11D.

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

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

Similarly, when compared with FIG. 11D, it can be seen that peaks corresponding to monosaccharides and disaccharides are observed in the test sample of Example 1 at the retention times of around 3.5 minutes and around 9 minutes, respectively. That is, it is considered that the test sample of Example 1 also contains such a monosaccharide or disaccharide component, whereby a high cell viability effect is obtained.

From the above results, in the polymer aqueous solution and the frozen solidified product of the present invention, DMSO, ethylene glycol, etc. can be produced by stably vitrifying the inside of the biological sample and preventing or suppressing the growth of ice crystals in the biological sample. Remarkable that biological samples can be cryopreserved with high cell viability without the need for addition of cell-permeable and cytotoxic compounds and / or serum or serum-derived proteins. It can be seen that it has a positive effect. The cells are well protected and their properties are maintained.

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

Further, the frozen solidified product of the present invention has an effect that it can be inferred whether or not the cells are well cryopreserved or not without thawing by the DSC measurement, the brightness measurement, and the measurement of the normal projection area of the cells. Also has.

Claims (17)

An aqueous polymer solution containing a water-soluble polymer or a salt thereof in an aqueous solvent and used for cryopreservation of biological samples.
The ultimate viscosity (η) of the aqueous polymer solution is 0.20 dL / g or more and 0.95 dL / g or less.
The normal projection area of the biological sample in the state where the polymer aqueous solution is solidified at −80 ° C. is reduced to less than 8/10 as compared with the normal projection area of the biological sample before solidification.
In the DSC curve of the temperature lowering process obtained by the following measurement conditions (1) and (2) using the differential scanning calorimeter (DSC) of the polymer aqueous solution, the calorific value of the exothermic peak in the temperature lowering process is 0J. A polymer aqueous solution characterized by having a calorific value of 65% or less of the corresponding calorific value of the reference liquid consisting of / g or water.
(DSC measurement conditions)
(1) After holding at 20 ° C for 1 minute, the temperature is lowered to -80 ° C at a temperature lowering rate of 5 ° C / min.
(2) After holding at -80 ° C for 1 minute, the temperature is raised to 20 ° C at a heating rate of 10 ° C / min. The normal projection area of the biological sample in the state where the polymer aqueous solution is solidified at −80 ° C. is reduced to a size of 1 / 3.5 or more as compared with the normal projection area of the biological sample before solidification. The polymer aqueous solution according to claim 1. In the DSC curve of the heating process obtained by the measurement conditions (1) and (2), the endothermic peak in the heating process is not confirmed, or the endothermic peak exceeds −1.4 ° C. 1 The polymer aqueous solution according to claim 1 or 2, which is at 1 ° C. or lower. 13. 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 a salt thereof in a content of 0.1 w / v% or more and 20 w / v% or less. The polymer aqueous solution according to any one of claims 1 to 5, wherein the water-soluble polymer is a water-soluble polymer containing a sugar residue. The polymer aqueous solution according to any one of claims 1 to 6, wherein the biological sample is a tissue-like substance such as a cell, a tissue, or a membrane or an aggregate. The polymer aqueous solution according to claim 7, wherein the biological sample is a mesenchymal stem cell, a blood cell cell, an endothelial cell, or a tissue for transplantation. The polymer aqueous solution according to claim 7, wherein the biological sample is a sperm, an egg, or a fertilized egg. The polymer aqueous solution according to any one of claims 1 to 9, which is used for vitrification and storage of the biological sample. A method for preventing or suppressing the growth of ice crystals in the biological sample during cryopreservation, wherein the biological sample is brought into contact with the aqueous polymer solution according to any one of claims 1 to 10. A solidified body containing a matrix and a biological sample containing an aqueous solvent and a water-soluble polymer or a salt thereof.
The biological sample has a reduced normal projection area of less than 8/10 as compared to before solidification.
When visible light is transmitted, the difference between the brightness in the Munsell color system of the region occupied by the biological sample and the brightness in the Munsell color system of the region occupied by the matrix is 3 or less when viewed from the light emitting side.
In the DSC curve of the temperature rising process obtained by the measurement conditions (1) and (2), the endothermic peak heat absorption in the temperature rising process is 0 J / g, or the corresponding heat absorption amount of the reference liquid consisting of water. A solidified body characterized by being 65% or less of.
(DSC measurement conditions)
(1) After holding at 20 ° C for 1 minute, the temperature is lowered to -80 ° C at a temperature lowering rate of 5 ° C / min.
(2) After holding at -80 ° C for 1 minute, the temperature is raised to 20 ° C at a heating rate of 10 ° C / min. The solidified body according to claim 12, which is a solid frozen product. The solidification according to claim 12 or 13, wherein the ultimate viscosity (η) of the aqueous polymer solution composed of the aqueous solvent and the water-soluble polymer or a salt thereof before solidification is 0.20 dL / g or more and 0.95 dL / g or less. body. The solidified product according to any one of claims 12 to 14, which contains the water-soluble polymer in a content of 0.1 to 20 w / v% or less. The solidified body according to any one of claims 12 to 15, wherein the biological sample is a tissue-like substance such as a cell, a tissue, or a membrane or an aggregate. The solidified body according to any one of claims 12 to 16, wherein the biological sample has a normal projection area reduced to a size of 1 / 3.5 or more as compared with that before solidification.

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