JP7350505B2 - Solidified bodies containing biological samples - Google Patents

Description

The present invention relates to a solidified body or a method for producing a solidified body containing a matrix and a biological sample.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The present invention has been made in view of the above problems, and provides a solidified body in which a biological sample is preserved at a high survival rate while retaining its properties. In particular, the solidified body contains a matrix containing water and a water-soluble polymer or a salt thereof together with the biological sample, which improves the cryopreservation effect of the biological sample, and furthermore, dimethyl sulfoxide (DMSO), propylene glycol, etc. It is safe because it does not contain chemicals such as (PG) and ethylene glycol (EG), nor does it contain serum or serum-derived proteins. Note that it is possible to include proteins that are not contaminated with bacteria or viruses. Furthermore, it is possible to add the above chemical substances at low concentrations that do not impair cell functions. The present invention also aims to provide a method for producing such a solidified body.

The present invention provides a solidified body containing a matrix containing an aqueous solvent and a water-soluble polymer or a salt thereof, and a biological sample, wherein the biological sample is The brightness in the Munsell color system of the area occupied by the biological sample when viewed from the light exit side when visible light is transmitted and has an orthogonal projected area reduced to 1/3.5 or more, and the matrix The difference in brightness in the Munsell color system of the area occupied by The present invention relates to a solidified material characterized in that no endothermic peak is observed in the DSC curve, or the temperature at which an endothermic peak is observed is greater than -1.4°C and 1.1°C or less. Here, the DSC scanning was performed by (1) holding at 20°C for 1 minute, then lowering the temperature to -80°C at a cooling rate of 5°C/min, (2) holding at -80°C for 1 minute, then lowering the temperature to -80°C at 10°C/min. The temperature is raised to 20° C. at a heating rate of min.

In this specification, "an endothermic peak is not observed" means that "an endothermic peak is not observed" unless an endothermic peak is observed in the range of -30°C to +5°C. If water containing a water-soluble polymer shows an endothermic peak, it is generally within this range. In addition, the orthogonal projected area of the biological sample, the area occupied by the biological sample, and the brightness in the Munsell color system of the matrix area remain unchanged after freezing and solidification. If the brightness of the Munsell color system in the matrix area has temperature dependence, the orthogonal projected area and the brightness of the Munsell color system are each measured at -80°C.

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

The solidified material of the present invention desirably does not contain dimethyl sulfoxide, which is an intracellular-penetrating cryoprotectant for biological samples. Further, it is preferable that the solidified material of the present invention does not contain a cryoprotectant such as ethylene glycol that is cytotoxic to biological samples. This is because these are harmful to the thawed biological sample.

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

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

The solidified material of the present invention has an endothermic amount of 0 J/g or Preferably, the amount of heat absorbed is 65% or less of the corresponding endothermic amount of the reference solution consisting of water.

Preferably, the biological sample contained in the solidified body is a cell, and the cells are mammalian cells.

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

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

The amount of HGF produced by the biological sample contained in the solution for biological sample administration in which the solidified body of the present invention is melted is lower than that of the biological sample in the solidified body containing DMSO instead of the water-soluble polymer or its salt. It is preferable that the amount of HGF produced is suppressed compared to the amount of HGF produced later.

The amount of IL-10 produced by the biological sample contained in the solution for biological sample administration in which the solidified body of the present invention is melted is determined by the biological sample in the solidified body containing DMSO instead of the water-soluble polymer or its salt. is preferably increased compared to the amount of IL-10 produced after thawing.

The present invention also provides a method for producing a solidified body containing a biological sample, in which a water-soluble polymer or a salt thereof is dissolved in an aqueous solvent and frozen at -80°C. It has an orthogonal projected area that is smaller than 1 and has been reduced to 1/3.5 or more compared to the culture solution (medium) before solidification, and when visible light is transmitted when frozen at -80°C. When viewed from the light exit side, the difference between the brightness in the Munsell color system of the area occupied by the biological sample and the brightness in the Munsell color system of the area occupied by the biological sample other than the area occupied by the biological sample is 3 or less, and the differential scanning calorimeter is In the DSC curve of the heating process obtained under the following measurement conditions (1) and (2) using (DSC), no endothermic peak is observed, or the temperature at which the endothermic peak is observed is -1.4°C. a step of preparing an aqueous polymer solution adjusted to have a property that the temperature exceeds 1.1°C, a step of including the biological sample in the aqueous polymer solution, and the aqueous polymer solution containing the biological sample. A method for producing a solidified body, comprising the steps of cooling and freezing the solidified body. Here, the DSC scanning was performed by (1) holding at 20°C for 1 minute, then lowering the temperature to -80°C at a cooling rate of 5°C/min, (2) holding at -80°C for 1 minute, then lowering the temperature to -80°C at 10°C/min. The temperature is raised to 20° C. at a heating rate of min.

In this specification, "an endothermic peak is not observed" means that "an endothermic peak is not observed" unless an endothermic peak is observed in the range of -30°C to +5°C. If water containing a water-soluble polymer shows an endothermic peak, it is generally within this range.

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

Further, it is desirable that the solidified material containing the biological sample of the present invention does not contain cell-permeable and cytotoxic compounds such as dimethyl sulfoxide and ethylene glycol. This is because these are toxic to cells after thawing. It is also desirable not to include serum or serum-derived proteins. Note that it is possible to add proteins that are not contaminated with bacteria or viruses. It is also possible to use cell-permeable and toxic compounds at low concentrations that do not impair cell function.

A method for producing a solidified material, wherein the step of cooling and freezing the aqueous polymer solution containing the biological sample is performed by cooling and freezing to a temperature of -27°C or less by a slow freezing method at a cooling rate of 10°C/min or less. is preferred.

In the method for producing a solidified material containing a biological sample according to the present invention, the temperature range for cooling and freezing the aqueous polymer solution containing a biological sample is not limited to -27°C or lower. The upper limit is desirably -70°C or lower, preferably -80°C or lower. The lower limit is desirably -196°C or higher, preferably -150°C or higher.

In the method for producing a solidified body containing a biological sample according to the present invention, the cooling rate when cooling and freezing the aqueous polymer solution containing a biological sample is preferably 10°C/min or less, preferably 1°C/min. It is preferable that the cooling and freezing steps are performed by a slow freezing method.

In the method for producing a solidified body containing a biological sample according to the present invention, it is preferable that the solidified body contains a water-soluble polymer or a salt thereof in a content of 0.1 w/v % or more and 20 w/v % or less.

In the method of producing a solidified body containing a biological sample of the present invention, a DSC curve of a heating process of an aqueous solution of a water-soluble polymer or its salt obtained under the above-mentioned DSC measurement conditions (1) and (2) is used. It is preferable that the endothermic amount of the endothermic peak in the temperature raising process is 0 J/g or 65% or less of the corresponding endothermic amount of the reference liquid consisting of water.

According to the present invention, biological samples such as cells whose properties are appropriately maintained without using cell-penetrating and toxic compounds such as intracellular-penetrating cryoprotectants such as DMSO and ethylene glycol can be used. A solidified body can be provided. The water-soluble polymer or its salt used in the present invention is non-intracellularly permeable and therefore has low toxicity to biological samples, yet exhibits a high cryoprotective effect on biological samples. , biological samples in solidified bodies are preserved with a high survival rate.

Furthermore, the solidified material of the present invention does not contain serum or serum-derived proteins to prevent damage to biological samples in the solidified material, and therefore is not contaminated with bacteria or viruses. Since there is no risk of containing unknown components or infectious substances derived from living organisms, the solidified product of the present invention can be used as it is as a solution for administering biological samples after being thawed. Therefore, it is considered suitable for storing biological samples for regenerative medicine applications, for example. Note that it is possible to use proteins that are not contaminated with bacteria or viruses. It is also possible to use cell-permeable and toxic compounds at low concentrations that do not impair cell function.

The water-soluble polymer or its salt forming the matrix of the solidified material of the present invention is dissolved in an aqueous solvent. Therefore, the matrix of the solidified material of the present invention may contain components derived from an aqueous solvent in addition to water and a water-soluble polymer or a salt thereof. Examples of aqueous solvents that can be used in the present invention include water, physiological solutions, and salt and sugar concentrations adjusted using sodium ions, potassium ions, calcium ions, etc. so that the osmotic pressure is approximately the same as the osmotic pressure of body fluids and cell fluids. Adjusted isotonic aqueous solutions are mentioned as preferred aqueous solvents. Specifically, the aqueous solvent includes, for example, water, physiological saline, phosphate buffered saline (PBS), which is a physiological saline with a buffering effect, Dulbecco's phosphate buffered saline, and Tris buffered saline. Physiological saline (Tris Buffered Saline; TBS), HEPES buffered saline, etc., balanced salt solutions such as Hank's balanced salt solution, Ringer's solution, lactated Ringer's solution, acetic Ringer's solution, bicarbonate Ringer's solution, 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 medium for various cells or tissues, commercially available medium, etc. It is. However, the aqueous solvent of the present invention preferably does not contain DMSO, ethylene glycol, serum, serum-derived proteins, or the like. Note that it is possible to add proteins that are not contaminated with bacteria or viruses. It is also possible to use cell-permeable and toxic compounds at low concentrations that do not impair cell function.

According to the solidified material of the present invention, biological samples such as cells can be mixed with water while maintaining the viability and properties of the biological sample without using cryoprotectants such as highly toxic DMSO or ethylene glycol. It can be cryopreserved in a matrix containing a water-soluble polymer or a salt thereof. The water-soluble polymer or its salt used in the present invention, which can function as a cryoprotectant in the solidified material of the present invention, promotes dehydration from the biological sample during freezing, thereby maintaining a good vitrification state of the biological sample. It can be realized both inside and outside of. The water-soluble polymer or its salt in the present invention is not cell membrane permeable. Therefore, when vitrifying a biological sample such as cells, the biological sample does not penetrate into the biological sample and swell the biological tissue. It has a reduced orthographic area compared to (in medium). In the water-soluble polymer of the present invention, by adjusting the molecular species and degree of polymerization (molecular weight) of the water-soluble polymer, and/or adding a low molecular weight or monomolecular compound to the aqueous solution of the water-soluble polymer. Accordingly, the temperature and amount of heat at the endothermic peak of melting during the heating process, as well as the difference in visible light transmittance between the solvent partial region of the solidified body and the biological sample internal region can be adjusted. In the present invention, by adjusting the DSC of the polymer aqueous solution so that no endothermic peak is observed or an endothermic peak appears near the endothermic peak of standard water (ultrapure water), and by adjusting the solvent partial region of the solidified body. The cryopreservation effect of living tissues such as cells can be improved by adjusting the difference in brightness when visible light is irradiated between the visible light and the internal region of the biological sample.

The solidified material of the present invention does not contain intracellular penetrating chemicals such as DMSO and ethylene glycol. Therefore, damage to biological samples, cytotoxicity, and effects on cell properties such as those reported for DMSO can be suppressed. Even after the solidified body is melted, the properties of the biological sample can be maintained appropriately.

Furthermore, the solidified material of the present invention can maintain the viability and properties of biological samples without containing serum and/or serum-derived proteins, so that biological samples are not contaminated with bacteria or viruses. Nor.

Furthermore, the solidified material of the present invention can be easily produced by slowly freezing an aqueous polymer solution containing a biological sample. In addition, the obtained solidified product can be stored at temperatures below -27°C, such as in a deep freezer, even though cytotoxic chemicals such as DMSO and ethylene glycol and proteins such as serum are not used. It can be stored stably for a long period of time. Note that it is possible to add proteins that are not contaminated with bacteria or viruses. It is also possible to use cell-permeable and toxic compounds at low concentrations that do not impair cell function.

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

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

The solidified body of the present invention is a solidified body that includes a matrix containing water and a water-soluble polymer or a salt thereof, and a biological sample. In the solidified body of the present invention, the biological sample has an orthogonal projected area that is smaller than 1 and reduced to 1/3.5 or more compared to before solidification.

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

In the solidified body of the present invention, the volume of the biological sample in the solidified body is reduced compared to that of the biological sample before freezing, as a result of accelerated discharge of water from within the biological sample during freezing and solidification. By effectively dehydrating the cells in this way, damage to the cells during freezing is significantly reduced, the cells are protected, and when the solidified material is thawed, the cells contained in the solidified material are removed. Biological samples show high viability.

In the solidified material of the present invention, the formation of ice crystals in the biological sample is suppressed or prevented by dehydrating the biological sample, and the matrix portion containing water and a water-soluble polymer or a salt thereof is combined with the biological sample. The inside of the sample is in a good vitrified state. Therefore, no ice crystals are formed either in the matrix portion or in the biological sample. When ice crystals with large crystal grains are formed, when visible light is transmitted through the solidified body, the visible light is blocked, and the brightness of the ice crystal portion decreases. In the solidified body of the present invention, the matrix portion of the solidified body and the inside of the biological sample are formed in a similar frozen state, that is, they are vitrified in the same way, so that the solidified body in the frozen state of the present invention has no visible When light is transmitted, the brightness inside the biological sample in the solidified body is close to the brightness in the matrix portion of the solidified body, that is, the area other than the inside of the biological sample, when viewed from the light exit side. Preferably, the difference between the brightness of the area occupied by the biological sample in the Munsell color system and the brightness of the area occupied by the matrix in the Munsell color system is 3 or less. Preferably, the frozen solidified matrix portion and the inside of the biological sample do not darken due to ice crystal formation.

Normally, in a freezing method called vitrification, when a solution is frozen, solutes (cryoprotectants to prevent frost damage) are removed from the crystals, resulting in an increase in the salt concentration in the remaining solution, which increases the concentration of salt inside and outside cells. This method dehydrates the inside of the cell and vitrifies the inside of the cell by creating an osmotic pressure difference, and is applied to cells whose survival rate after thawing is particularly low. In such a method, in order to make water more easily vitrified, the concentration of a solute (cryoprotectant) is increased or the cooling rate is increased. However, it has been known that increasing the osmotic pressure difference causes greater damage to cells, and that recrystallization occurs during dissolution, resulting in cell damage. It is also technically difficult.

In the present invention, dehydration from a biological sample and its promotion are realized by a matrix containing water and a water-soluble polymer or a salt thereof. The matrix of the present invention can effectively trap aqueous solvent molecules within the matrix formed by polymer chains during the cooling process. This restricts the molecular movement of the water in the aqueous solvent upon cooling, allowing the water to solidify and/or freeze in a vitrified state without crystallizing, ie without forming ice crystals.

During solidification and freezing, the water-soluble polymer used in the present invention forms an amorphous glassy matrix due to the action of its polymer chains. In the solidified product of the present invention, the water molecules of the solvent are thought to exist in the network matrix of the polymer, and exist in the state of free water or bound water (a state in which water molecules are entangled and captured by the polymer). It will be done. Furthermore, it is thought that water molecules that have been removed from cells etc. due to the osmotic pressure difference during solidification and freezing are replaced with macromolecules surrounding the biological sample such as cells. This substitution may further facilitate the movement of water molecules from within the biological sample to the outside. As a result, the formation of ice crystals within the biological sample is suppressed and prevented, and the inside of the biological sample becomes vitrified.

In other words, in the solidified material of the present invention, both the inside and outside of the biological sample are vitrified by the action of the polymer chains of the water-soluble polymer, so the osmotic pressure in the biological sample during freezing, which is a problem with conventional vitrification methods, is reduced. Shock can be weakened. Therefore, there is no need to contain cytotoxic chemical substances such as dimethyl sulfoxide (DMSO) and ethylene glycol, and the solidified material of the present invention does not contain DMSO and/or ethylene glycol.

The solidified material of the present invention contains a matrix having such excellent vitrification ability. In such a matrix of the present invention, the endothermic peak measured by DSC in the DSC curve of the temperature rising process obtained under the following measurement conditions (1) and (2) using a differential scanning calorimeter (DSC) is Either no endothermic peak is observed, or the temperature at which an endothermic peak is observed is greater than -1.4°C and 1.1°C or less.
DSC measurement conditions:
(1) After holding at 20°C for 1 minute, lower the temperature to -80°C at a cooling rate of 5°C/min, and (2) After holding at -80°C for 1 minute, lower the temperature to 20°C at a heating rate of 10°C/min. Temperature rising.

That is, in the matrix of the present invention, the endothermic peak estimated to be caused by the melting of water is not observed during the heating process of the matrix, or if it is observed, it exceeds -1.4°C, and 1. It exists in the range of 1° C. or lower, that is, near the temperature (0.7° C.) at which an endothermic peak is observed in ultrapure water under DSC measurement under the same conditions. In order to vitrify a solvent or a biological sample such as a cell, it generally seems desirable to use a solution having a lower melting point (freezing point). However, the present inventors have found that for good vitrification of biological samples upon freezing, it is desirable for the matrix to have characteristics opposite to this common knowledge. Normally, the phenomenon that the melting point (freezing point) is lower than that of water occurs when a solute is dispersed in a water matrix, but in the matrix containing water-soluble polymers in the present invention, the opposite phenomenon occurs. Water molecules exist in a polymer network matrix in an aqueous solvent formed of water-soluble polymers, and water exists as free water without freezing in the polymer matrix, or water Even if it freezes, it freezes when it is entangled in polymers (bound water). Therefore, in the aqueous polymer solution of the present invention, it is presumed that the endothermic peak of water melting is not observed during the temperature raising process, or that it is observed at a temperature close to the temperature at which the endothermic peak of pure water is observed.

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

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

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

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

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

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

In order for water molecules to be efficiently trapped in the matrix formed by the polymer chains of the water-soluble polymer in the present invention during the cooling process, in the present invention, for example, the water-soluble polymer or its salt is added to the aqueous solvent. The intrinsic viscosity of the dissolved polymer aqueous solution can be adjusted to about 0.20 to 0.95 dL/g. If the intrinsic viscosity of the aqueous polymer solution is too small, the polymer will diffuse, making it impossible to entangle and capture water molecules in the water-soluble polymer network. On the other hand, if the intrinsic viscosity of the aqueous polymer solution is too high, replacement with water molecules near the biological sample will not proceed. Further, there may arise a problem that handling properties deteriorate when a biological sample is included in a polymer solution for cryopreservation. By setting the intrinsic viscosity of the polymer aqueous solution within the range of 0.20 dL/g or more and 0.95 dL/g or less, when the polymer aqueous solution is cooled, the amorphous glass state in the frozen state is stabilized. can be converted into Therefore, it is thought that the biological sample is not easily damaged by cooling and freezing, and the biological sample is efficiently stored in the solidified body while maintaining stable high viability. Destruction of cells in biological samples due to ice crystal formation is also less likely to occur. Therefore, the survival rate of cells in the biological sample in the solution after melting the solidified material of the present invention is high.

Note that the "intrinsic viscosity (η)" of the aqueous solution of 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水溶液の極限粘度とする。 Intrinsic viscosity measurement:
(1) A predetermined amount of NaCl is dissolved in ion-exchanged water at 30°C to prepare a 0.2M NaCl solution (standard solution).
(2) Dissolve a sample of a water-soluble polymer or its salt in a standard solution at 30°C to prepare a stock solution. When a sample of a water-soluble polymer or its salt is obtained in the form of a solution, the solid content obtained by removing the solvent from the solution is used as the sample of the water-soluble polymer or its salt. In the case of a mixed sample containing multiple water-soluble polymers, use the sample as is. Even if it is a mixture of water-soluble polymers mixed with impurities, it can be used as a sample. However, this is not the case when measuring the viscosity average molecular weight described below. In the case of a mixed sample containing a plurality of water-soluble polymers, each water-soluble polymer is separated and fractionated, and then the solvent is removed from each solution to obtain a sample of each water-soluble polymer. If the water-soluble polymer is unknown, the substance is identified by HPLC, LC-MS, LC-IR, etc., and the viscosity average molecular weight is calculated as described below. In addition, if multiple unknown water-soluble polymers are included, separate and fractionate each component, identify the substance of each water-soluble polymer by HPLC, LC-MS, LC-IR, etc., and proceed as described below. Calculate the viscosity average molecular weight. Note that even if the mixture contains impurities in the water-soluble polymer, if the viscosity is not affected (for example, impurities such as metal salts), the mixture is used as a sample of the water-soluble polymer. Furthermore, if the sample contains impurities that affect the calculation of the viscosity average molecular weight, the impurities are removed or the water-soluble polymer is fractionated before measurement. At this time, the viscosity of each of the standard solution and stock solution is measured, and the relative viscosity of the stock solution with respect to the standard solution is adjusted to be 2.0 to 2.4.
(3) Dilute the 30°C stock solution to 5/4, 5/3, and 5/2 times using the 30°C standard solution.
(4) Measure the viscosity of the standard solution, stock solution, and diluted solution at 30°C. An E-type viscometer is used for viscosity measurement.
(5) The relative viscosity (η _r ) is obtained by dividing the viscosity of each of the stock solution and diluted solution by the viscosity of the standard solution, and the reduced viscosity is derived based on the following formula.

Here, η _sp : Reduced viscosity of the water-soluble polymer or its salt [mL/g], η _r : Relative viscosity of the water-soluble polymer or its salt [-], C: Concentration of the water-soluble polymer or its salt [g] /mL].
(6) Plot the relationship between the concentration of the water-soluble polymer or its salt and the reduced viscosity of the water-soluble polymer or its salt, and draw an approximate straight line. The value of the intercept of the approximate straight line (polymer or saccharide concentration = 0) is taken as the limiting viscosity of the aqueous solution of the water-soluble polymer or its salt.

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

For example, the water-soluble polymer or its salt used in the present invention is, for example, a water-soluble polymer or its salt having a viscosity average molecular weight of more than 3,000 and less than 500,000. By using a water-soluble polymer or its salt with a viscosity average molecular weight of this level, when a matrix containing a water-soluble polymer is cooled, the amorphous glass state in the frozen state is stabilized. Ru. Therefore, the cells are protected by the matrix during cooling and freezing, and as a result, the cells are less likely to be damaged, and the cells can be stably and efficiently cryopreserved in the solidified body. Therefore, the survival rate of cells in the biological sample after thawing the solidified material after cryopreservation is high. If the viscosity average molecular weight of the water-soluble polymer is 3000 or less, vitrification may be difficult to occur satisfactorily. Furthermore, if the viscosity average molecular weight of the water-soluble polymer is greater than 500,000, the viscosity will increase significantly, and the solubility may decrease, and the prepared solution may foam, resulting in poor handling properties. For example, the viscosity average molecular weight is preferably 10,000 or more. Further, a polymer having a viscosity average molecular weight of 400,000 or less, more preferably 200,000 or less is preferable, and 150,000 or less is particularly preferable. This is because the viscosity can be adjusted to be low as an aqueous solution and it is easy to handle.

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

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

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

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

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

The hydrophilic groups of the water-soluble polymer or its salt used in the present invention are either unmodified or, even if modified, 50% or less of the total number of hydrophilic groups, that is, substituents are introduced into the polymer chain. It is desirable that the number of hydrophilic groups is not more than 50% of the total number of hydrophilic groups. It is assumed that the hydrophilic groups of water-soluble polymers, especially OH groups, NH ₂ groups, and COOH groups, contribute to the protection of biological samples, vitrification of the matrix, and replacement with water molecules around biological samples. This is because it is advantageous to improve the survival rate of biological samples if these functional groups are not modified. Furthermore, when attempting to introduce functional groups into water-soluble polymers, the reagents used to modify the hydrophilic groups of the polymers may adversely affect the cryoprotective effect of the polymers or There is a risk that it may become unusable as a cryopreservation solution for cryopreservation of samples.

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

The low molecular weight saccharide or its salt is held within the polymer matrix by hydrogen bonding by hydrophilic groups in the water-soluble polymer. As a result, it is thought that the amount and balance of water that exists as free water and bound water trapped in the polymer matrix changes. Furthermore, low molecular weight saccharides or salts thereof are thought to have the effect of further promoting dehydration within the biological sample during the freezing process. That is, it is presumed that when a polymer holding a low molecular weight saccharide or a salt thereof is present around a biological sample such as a cell, water molecules near the cell membrane are also replaced with the low molecular weight saccharide. Therefore, it is thought that water discharged from a biological sample such as cells during freezing due to the osmotic pressure difference replaces not only the macromolecules surrounding the biological sample such as cells but also low molecular weight sugars. Therefore, the addition of low molecular weight sugars or their salts is believed to aid in the drainage of water from within the biological sample and, through replacement with water molecules, suppress or prevent the formation and growth of ice crystals near cell membranes. For example, by setting the intrinsic viscosity of the aqueous polymer solution to a range of 0.20 dL/g or more and 0.95 dL/g or less, replacement of water molecules and low molecular weight saccharides can be promoted. As a result, cell membrane damage during freezing can be significantly suppressed. In other words, a low molecular weight saccharide or a salt thereof can function as a component for cell protection in an aqueous polymer solution. If the intrinsic viscosity of the aqueous polymer solution is too small, low molecular weight saccharides will diffuse into the polymer solution, and water molecules may not be efficiently entangled and captured in the water-soluble polymer network. On the other hand, if the intrinsic viscosity of the aqueous polymer solution is too high, it is thought that the replacement of low molecular weight sugars with water molecules will not proceed efficiently.

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

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

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

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

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

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

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

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

On the other hand, the water-soluble polymer or its salt used in the present invention is, for example, hyaluronic acid with a viscosity average molecular weight of 15,000 and an endothermic peak temperature of -1.4°C, pullulan with a viscosity average molecular weight of 373,000, and 1. The temperature is 49°C. According to the present invention, when such a water-soluble polymer or its salt is combined with a low molecular weight saccharide or its salt, the endothermic peak temperature of the aqueous polymer solution is brought close to that of pure water, or the endothermic peak temperature is itself is no longer observed in DSC measurements. For example, when hyaluronic acid with a viscosity average molecular weight of 1,000 or less is added to hyaluronic acid with a viscosity average molecular weight of 15,000, the endothermic peak temperature does not decrease, but increases to -0.4°C. On the other hand, when hyaluronic acid with a viscosity average molecular weight of 1,000 or less is added to pullulan with a viscosity average molecular weight of 373,000, the endothermic peak temperature decreases to 0.83°C.

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

The low molecular weight saccharides or salts thereof that exhibit such effects are not limited to the above-mentioned hyaluronic acid having a viscosity average molecular weight of 1000 or less. As long as the viscosity average molecular weight is 1000 or less, the same effect can be achieved not only with hyaluronic acid but also with other saccharides such as sucrose and trihalose. During the freezing and solidification process of the matrix of the present invention, low molecular weight sugars either exist between water molecules and inhibit the freezing of water, or work together with a network of water-soluble polymers to transport water within the network. By trapping (in either bound water or free water state) and inhibiting the ice crystallization of water in the first place, the aqueous polymer solution of the present invention acts to achieve behavior close to that of pure water. It is speculated that this may be the case.

Therefore, in the solidified material of the present invention, the temperature at which an endothermic peak appears can be adjusted by adding a compound such as a low molecular weight saccharide to the water-soluble polymer. This shows that it is possible to adjust the trap state of water molecules within the polymer network by bringing the temperature close to the endothermic peak temperature of water or by preventing the endothermic peak from appearing. Therefore, in the solidified body of the present invention, the protective effect for the biological sample contained in the solidified body is adjusted to be appropriate.

Furthermore, in the solidified material of the present invention, by adding compounds such as low molecular weight sugars to the water-soluble polymer, the brightness in the Munsell color system of the area occupied by the biological sample in the solidified material and the matrix in the solidified material can be adjusted. It is possible to adjust the difference between the brightness of the area occupied by the area and the brightness in the Munsell color system to 3 or less. This indicates that the degree of ice crystal formation, that is, the degree of vitrification, within a biological sample can be adjusted by adding compounds such as low molecular weight saccharides. Therefore, the solidified body of the present invention is adjusted so that both the inside of the biological sample and the outside of the biological sample (matrix region) in the solidified body can be in a good vitrification state.

As a result, in the present invention, water does not form ice crystals in solid frozen materials containing biological samples obtained after freezing, and therefore biological samples such as cells are not destroyed by ice crystals, resulting in a high survival rate. It is stored in

In the solidified material of the present invention, since no ice crystals are formed in the biological sample region, visible light is not blocked by ice crystals. As mentioned above, the visible light transmittance of the solvent part in the solidified body and the biological sample area is almost the same, and even when observed under a microscope, the difference in brightness between the solvent part and the biological sample area is according to the Munsell color chart. It is 3 or less in the system. Therefore, simply by irradiating the frozen solidified body with visible light and observing it under a microscope, it is possible to easily confirm whether or not the biological sample in the solidified body is alive.

The solidified material of the present invention contains a water-soluble polymer or a salt thereof at a concentration of approximately 0.1 w/v% or more and 20 w/v% or less. If the concentration of the water-soluble polymer is too low, the solvent portion may not be vitrified satisfactorily. For example, the concentration of the water-soluble polymer or its salt is preferably 1 w/v% or more, particularly preferably 5 w/v% or more. Furthermore, if the concentration is higher than 20 w/v%, the viscosity may become too high and the handling properties may deteriorate. Preferably, the concentration of the polymer or its salt is 5 w/v% or more and 20 w/v% or less.

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

Further, the matrix of the present invention preferably contains a low molecular weight saccharide or a salt thereof in addition to the water-soluble polymer. As mentioned above, the amount of low molecular weight saccharide or its salt added is such that the difference between the brightness of the area occupied by the biological sample in the solidified body and the brightness of the area occupied by the matrix in the solidified body is smaller than the desired value. and/or the temperature at which the endothermic peak appears may be adjusted as appropriate to bring it close to the desired temperature; however, for example, addition of low molecular weight sugars or salts thereof may The content ratio of the molecular weight saccharide to the aqueous polymer solution may be about 10:1. If the concentration of the saccharide or its salt is too low, the cell protection effect of the low molecular weight saccharide may not be sufficiently obtained. Further, even if saccharides are added at a concentration of 1/10 times or more that of the water-soluble polymer, it is difficult to obtain further effects as a cell-protecting component.

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

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

According to the results of thermal analysis of the matrix of the present invention by differential scanning calorimetry (DSC), in the matrix of the present invention, in the DSC curve of the heating process obtained under the following measurement conditions (1) and (2), , the endothermic amount of the endothermic peak in the temperature raising process is 0 J/g or 65% or less of the corresponding endothermic amount of the reference solution consisting of water, preferably, the corresponding endothermic amount of the reference solution consisting of water. It is 40% or less.

The fact that the endothermic amount of the endothermic peak in the temperature raising process in the DSC measurement is smaller (less than 100%) than the corresponding endothermic amount of the reference solution consisting of water in the matrix of the present invention means that ice crystals in the frozen matrix This means that there remains bound water and free water that do not form. That is, in the aqueous solution of the water-soluble polymer forming the matrix of the present invention, the water-soluble polymer and/or low molecular weight saccharides are replaced with water, and water molecules are trapped within the matrix of the water-soluble polymer. This restricts the movement of water molecules. This promotes the dehydration of water from within the cells of the biological sample contained in the matrix, and also allows the water to be solidified in a glassy state without crystallizing. Good vitrification of the matrix in the solidified body and the inside of the biological sample contained in the solidified body is achieved. Therefore, in the solidified product of the present invention, destruction of cells due to the formation of ice crystals can be suppressed or prevented. Furthermore, in the solidified body of the present invention, not only the formation of ice crystals is suppressed or prevented during the cooling process, but also the recrystallization of water during the subsequent temperature raising process, that is, during melting. That is, in the solidified body of the present invention, it is considered that the glass state of the biological sample contained in the solidified body in the frozen state is stabilized. Since the glass state is further stabilized in this way, biological samples stored in the solidified body of the present invention do not require the addition of protein components such as human or bovine serum or serum-derived components (such as albumin). It shows a high cytoprotective effect without any damage. Therefore, the thawed biological sample is free from infectious diseases and is not affected by lot-to-lot differences due to biological preparations. Note that it is possible to add proteins that are not contaminated with bacteria or viruses.

Since the solidified material of the present invention does not contain a cell-permeable compound, it is considered to have low cytotoxicity when used for cryopreservation of biological samples. Furthermore, since there is no need to increase the concentration of solutes to limit the movement of water molecules, damage to cells is thought to be low. In addition, the added low molecular weight saccharide or its salt used in the present invention has a cytoprotective effect that rapidly replaces the water excreted from inside the cell around the cell during freezing, so it protects the cell during cryopreservation. It is thought that it does not change the properties of Therefore, it is considered that biological samples are cryopreserved in the solidified body of the present invention while maintaining cell characteristics.

Furthermore, unlike conventional vitrification methods, the solidified product of the present invention does not require a large cooling rate during cooling to reduce osmotic shock during production. Therefore, according to the solidified material of the present invention, biological samples can be efficiently cryopreserved in a simple method with reduced toxicity, and when the solidified material is thawed after cryopreservation, The biospecimens show a high survival rate.

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

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

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

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

That is, by including a biological sample selected from cells, tissues, tissue-like substances such as membranes or aggregates in the solidified body of the present invention, a high survival rate can be achieved in cryopreservation of biological samples. I can do it. In particular, the matrix containing the water-soluble polymer of the present invention can be applied to mesenchymal stem cells, hematopoietic stem cells, neural stem cells, somatic stem cells such as bone marrow stem cells and germline stem cells, and blood cells, regardless of whether they are primary cells or established cells. Advantageous in the cryopreservation of endothelial cells, etc., especially the cryopreservation of primate stem cells, which are said to have lower freezing tolerance than mice, the cryopreservation of tissues for transplantation, and the cryopreservation of germ cells in reproductive medicine. can be used.

Further, if a water-soluble polymer which is a biological component is used as the water-soluble polymer in the present invention, for example, it is possible to melt the solidified material and use it as it is as a solution for administering a biological sample. Furthermore, in the case of tissue or tissue-like material, it is also possible to add a matrix containing such a water-soluble polymer of the present invention at the stage of organization and freeze it as it is to form a solidified product. This is thought to reduce post-transplant problems. In such cases, the preferred water-soluble polymer for the water-soluble polymer-containing matrix of the present invention is hyaluronic acid. In particular, hyaluronic acid has a viscosity average molecular weight of greater than 3,000, more preferably greater than 5,000, and less than 60,000, more preferably less than 20,000.

The method of producing a solidified body containing a biological sample according to the present invention involves dissolving a water-soluble polymer or its salt in an aqueous solvent, and performing the above-mentioned method, that is, when the biological sample is frozen at -80°C, the biological sample is frozen. , has an orthogonal projected area that is smaller than 1 and reduced to 1/3.5 or more compared to the culture solution (medium) before solidification, and when visible light is transmitted when frozen at -80°C When viewed from the light exit side, the difference between the brightness in the Munsell color system of the area occupied by the biological sample and the brightness in the Munsell color system of the area other than the area occupied by the biological sample is 3 or less, and the differential scanning calorimeter (DSC) ) In the DSC curve of the heating process obtained under the measurement conditions (1) and (2) below, no endothermic peak is observed, or the temperature at which the endothermic peak is observed exceeds -1.4°C, A step of preparing an aqueous polymer solution adjusted to have a temperature of 1.1°C or less, a step of including a biological sample in the aqueous polymer solution, and a step of cooling and freezing the aqueous polymer solution containing the biological sample. It includes the process.
Here, DSC measurement conditions:
(1) After holding at 20°C for 1 minute, lower the temperature to -80°C at a cooling rate of 5°C/min, and (2) After holding at -80°C for 1 minute, lower the temperature to 20°C at a heating rate of 10°C/min. Temperature rising.

Since the aqueous solution of the water-soluble polymer that forms the matrix of the present invention has a glass transition temperature around -23°C ± 4°C, the vitrification of the matrix is caused by the formation of a network of water-soluble polymers during freezing. This is achieved by entangling and trapping water molecules, so the process for producing the solidified material of the present invention does not require a large cooling rate. For this reason, for example, the aqueous polymer solution of the present invention containing a biological sample is cooled to −27° C. or lower, which is the glass transition point temperature of the aqueous polymer solution, after the biological sample is included in the aqueous polymer solution used in the present invention. Cells and tissues to be preserved can be stably frozen in a solidified form while maintaining their viability by simply placing them in a freezing treatment container and leaving them in a -80°C deep freezer. can do.

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

Cooling and freezing in the process of manufacturing the solidified product of the present invention is performed by a slow freezing method. That is, in order to produce the solidified product of the present invention, the cooling rate is preferably 10° C./min or less. More preferably, the cooling rate is about 1° C./min or less. It is considered that at this cooling rate, the inside of the biological sample is appropriately dehydrated, the liquid within the biological sample is vitrified well, and a high cell cryoprotection effect is obtained.

In the solidified material of the present invention, the vitrification state is stabilized and the toxicity to biological samples is low, so that biological samples can be stably stored for a long period of time. In this specification, long-term stable storage means, for example, that after the solidified material of the present invention is thawed, the survival rate of a biological sample, such as cells, is 5 months based on the cell viability immediately before storage. After that, there is a decrease of less than 10%, preferably less than 5%, or after 6 months, there is a decrease of less than 20%, preferably less than 10%, or after 12 months, it is less than 15%, preferably means that a decrease of less than 30% is observed. Furthermore, in this specification, long-term stable storage means, for example, storing cells in a solidified body at -80°C for a long period of time, thawing the solidified body, and then storing the cells as they are at 4°C. In this case, even 24 hours after thawing, it means that the viability is reduced by less than 5% based on the cell viability immediately after thawing. In the solidified material of the present invention, cells are considered to be cryopreserved under conditions with less stress than in conventional cryopreservation methods that use a DMSO solution as a cryopreservation solution. Therefore, with the biological sample preserved as a solidified material of the present invention, a significantly higher cell survival rate after thawing can be obtained compared to a cryopreservation method using a conventional cryopreservation solution such as a 10% DMSO solution. Can be done. Furthermore, not only cells immediately after thawing the solidified material but also cells refrigerated after thawing can exhibit a high cell survival rate. In the solidified product of the present invention, cells can be stably cryopreserved for a long period of time without changing the properties of the cells. Therefore, it is suitable for storing biological samples for regenerative medicine applications, for example. Since no special techniques or equipment are required to manufacture and preserve the solidified material of the present invention, the solidified material of the present invention containing a biological sample with a high survival rate can be easily produced, and its transportation etc. It is easy and can facilitate the handling of biological samples.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

・Example 3
A test at a pullulan concentration of 10 w/v% in which 1 g of the test sample of Comparative Example 9 (pullulan with a viscosity average molecular weight of 373,000) was dissolved in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent is water). The test sample of Production Example 1 (hyaluronic acid fragment sample with a viscosity average molecular weight of 1000) was added to the test sample solution of Example 3 at a final concentration of 1 w/v%. The intrinsic viscosity was 0.55 dL/g.

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

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

Sucrose (Nacalai Tesque Co., Ltd.) is added to a test sample solution with a hyaluronic acid concentration of 10 w/v% in which 1 g of this hyaluronic acid is dissolved in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent is water). ), molecular weight (equivalent to viscosity average molecular weight) is 342) and glucuronic acid (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd., molecular weight (viscosity average molecular weight and fictitious) 194) at a final concentration of 1 w/v%. The cryopreservation solution for the test of Example 5 was obtained by adding it. The intrinsic viscosity was 0.32 dL/g.

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

Thereafter, the cell suspension containing each test sample solution was frozen in a -80°C freezer in a slow cell freezer (Nalgene® Mr. Frosty) to obtain a solidified body. After storing the solidified product at -80°C for 7 days, it was rapidly thawed in a 37°C bath. Immediately after thawing, the cell suspension containing each test sample solution was evaluated for cell viability by trypan blue staining. The results are shown in FIG. 1 and Table 1 (Table 1 only shows the results of Example 5).

Figure 1 shows a medium without cryopreservation solution, that is, a solidified body containing the test sample solution of Comparative Example 2, or a solidified body containing the test sample solution of Example 1 and Comparative Examples 1, 4, 5, 7, and 8. It shows the cell survival rate after thawing of cells cryopreserved in a solidified material containing the same. As shown in FIG. 1, in Example 1, that is, the hyaluronic acid obtained by subcritical treatment has a viscosity average molecular weight of about 10,000, and further has a viscosity average molecular weight of 1000, that is, smaller than 10,000. A solidified body containing the test sample solution of Example 1, wherein the hyaluronic acid sample containing a cleavage product of hyaluronic acid having a molecular weight is a water-soluble polymer, and the aqueous solution has an intrinsic viscosity of 0.49 dL/g. A significantly high preservation effect of over 90% was obtained. This preservation effect was even higher than that of Comparative Example 1, which contained DMSO, which is a commonly used cryopreservative, as a cryopreservative. It can be seen that freezing in the solidified material of the present invention is an excellent cryopreservation with low cytotoxicity.

In the frozen solidified product of Example 1, no endothermic peak was observed in the DSC measurement during the heating process (see Test Example 2 below), water molecules did not form ice crystals, and free water or hyaluronic acid molecular chains did not form. It is estimated that the water is adsorbed or entangled and becomes bound water. In addition, since the difference in brightness between the solvent matrix region and the cell region of this freeze-solidified product is as small as 2 (see Test Example 8 below), the state of water molecules in the solvent matrix region and the cell region are similar, and the intracellular state of water molecules is similar. It is also thought that no ice crystals are formed. Furthermore, when the test sample solution was solidified at -80°C, the area of the cells shrank to 38% of the area of the cells in the culture solution (medium) before solidification (see Test Example 9 below). (see), it is thought that hyaluronic acid does not invade into cells, and that water molecules move out of cells due to osmotic pressure differences. Water molecules that move outside the cell are thought to be replaced with low-molecular-weight hyaluronic acid or high-molecular-weight hyaluronic acid, and may remain around cells and form ice crystals, causing problems such as destruction of cell tissue. Furthermore, hyaluronic acid does not enter cells and harm them. Therefore, unlike DMSO in Comparative Example 1, it has low cytotoxicity and does not form ice crystals in or around cells, so it is also excellent in cell cryopreservation effects.

In the solidified body containing the test sample solution of Comparative Example 4, which contained high molecular weight hyaluronic acid with a high molecular weight of 1,000,000 and had a high intrinsic viscosity of 17.2 dL/g, almost no cell protection effect was observed. In the freeze-solidified product of Comparative Example 4, no endothermic peak was observed in the DSC measurement during the heating process (see Test Example 2 below), but no contraction was observed in the freeze-solidified cells, and water molecules 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), and the cell region is darkened, indicating that the water molecules in the cell are frozen. It is thought that crystals are formed. For this reason, it is presumed that the ice crystals destroyed the cells and no effect on cell preservation was observed.

Note that in Comparative Example 2, which did not contain a cryopreservative, the cell survival rate was approximately 0%.

Furthermore, in Comparative Example 7, which contained carboxypolylysine as a test sample and had a small intrinsic viscosity of 0.11 dL/g, no cell protection effect was observed as in Example 1 or Comparative Example 1 in which DMSO was used as a cryoprotectant. In Comparative Example 7, the temperature at which the endothermic peak appeared in the DSC measurement during the heating process was as low as -9.66°C (see Test Example 2 below), and the freezing rate of water was slow, causing ice crystals to form. Conceivable. In addition, since the difference in brightness between the solvent matrix region and the cell region is small (see Test Example 8 below), the ice crystal formation state in the solvent matrix region and the cell region are similar, and ice crystals are also formed within the cells. Furthermore, since the area of the cells after freezing is larger than the area in the culture solution (medium) (see Test Example 9 below), it is likely that polylysine has invaded the cells. It is thought that the cells are expanding due to ice crystals. It is speculated that this may damage the cells and prevent a sufficient preservation effect from being obtained.

Regarding the fructan of Comparative Example 8, the temperature at which the endothermic peak appears is as low as -2°C (see Test Example 2 below), and it is thought that the freezing rate of water is slow and that ice crystals are likely to form. The area was larger than the area in the culture solution (medium), and rupture was also observed (see Test Example 9 below), suggesting that fructans may have entered the cells. In addition, the difference in brightness between the solvent matrix area and the cell area after freezing and solidification was as large as 5, and the cell area had turned dark (see Test Example 8 below), indicating that ice crystals were formed within the cells. Therefore, it seems that sufficient preservation effect cannot be obtained.

In this way, the endothermic peak in DSC measurement during the temperature rising process does not appear, or the temperature at which it appears is set to appear around 0.7°C (-1.4°C to 1.1°C), which is the endothermic peak of water. This adjustment suppresses the formation of ice crystals in the solvent matrix, reduces the difference in brightness between the solvent matrix region and the cell region, approximates the state of ice crystal formation in both regions, and solidifies the cells so as to shrink them. This suppresses the entry of water-soluble polymers into cells, reduces intracellular water molecules, and prevents the formation of intracellular ice crystals, resulting in excellent cell cryopreservation effects. It is believed that this can be achieved.

Furthermore, the frozen solidified product of the present invention has the effect that it is possible to estimate whether or not the cells have been well cryopreserved by DSC measurement, brightness measurement, and measurement of the orthogonal projection area of the cells without thawing them. have.

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

Furthermore, the solidified material of the present invention showed a high cell preservation effect even when produced using a culture medium such as αMEM medium instead of water as an aqueous solvent for dissolving the water-soluble polymer. The solidified product can be produced by adding the aqueous polymer solution as it is to the cell culture solution, suspending the cells, and then freezing the cells, and can be stored as is. It is considered that there is no need to centrifuge the cells to be stored, and it is possible to maintain higher cell survival rates and cell properties.

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

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

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

As shown in Figures 2A and 2B, in Comparative Example 2 where the test sample was ultrapure water (i.e., the sample was ultrapure water), only the ice crystal melting peak of free water was observed (Figure 2A ). In Comparative Example 5, in which the test sample was hyaluronic acid with a viscosity average molecular weight of 15,000, a shift in the ice crystal melting peak and a drop in the freezing point were observed compared to Comparative Example 2 (Figure 2A), and the temperature of the glass at around -20°C was observed. Metastasis was confirmed (Fig. 2B). On the other hand, in the test sample of Example 1 containing hyaluronic acid with a viscosity average molecular weight of about 10,000 and a cleavage product of hyaluronic acid (low molecular weight hyaluronic acid with a viscosity average molecular weight of 1000), the peak of ice crystal melting was It was extremely small, almost not observed (FIG. 2A), and only a glass transition was observed around -23°C±4°C (FIG. 2B). This result shows that in the solidified material of Example 1, water exists in the matrix in a state that does not freeze (free water) or in a state that is entangled with polymers and is trapped (bound water), and that This shows the significantly advantageous effect of the solidified product of the present invention in that the recrystallization of water is suppressed.

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

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

On the other hand, the endothermic peak appearance temperature of Comparative Example 9 (the test sample was pullulan with a viscosity average molecular weight of 373,000) was 1.49°C, which was higher than the endothermic peak appearance temperature of pure water, which was 0.7°C. When this Comparative Example 9 is mixed with Production Example 1 (endothermic peak appearance temperature is -8.1°C) (Examples 3 and 4), the endothermic peak appearance temperature is lowered rather than increased, and the pure water The endothermic peak appearance temperature was 0.83°C, which is close to 0.7°C.

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

On the other hand, the endothermic peak temperature of the hyaluronic acid fragment having a viscosity average molecular weight of 2000 or less in Production Example 2 was -0.3°C as shown in Table 1. The endothermic peak appearance temperature of Comparative Example 10, in which the low molecular weight sample of Production Example 2 was mixed with Comparative Example 5 (endothermic peak appearance temperature is -1.4°C), was lowered to -1.6°C.

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

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

The endothermic peak temperatures of the test sample solutions of Examples 1 to 5 are around 0.7°C, the endothermic peak temperature of water, that is, they exhibit behavior similar to pure water, and the endothermic amount of the endothermic peak is similar to that of water. The fact that the endothermic peak observed during the temperature increase process is smaller than the endothermic amount indicates that bound water and free water that do not form ice crystals remain in the matrix in the frozen solidified material of the present invention. It shows. It is thought that the water-soluble polymer used in the present invention achieves vitrification by trapping water molecules within its matrix upon freezing.

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

The results shown in FIG. 4A show that the cells frozen in the solidified body of the present invention exhibit a high cell survival rate of 95% or more, which remains almost unchanged even after 5 months of storage. On the other hand, in the cryopreservation using the sample solution containing the test sample (DMSO) of Comparative Example 1, a decrease in cell viability was already observed after two months of cryopreservation. It can be seen that after 3 months, the cell survival rate in the cryopreservation of Comparative Example 1 was below 50%. This result was in contrast to the solidified material of the present invention, which still maintained a high cell survival rate even after 3 months. After 6 months of cryopreservation, cell viability decreased by less than 10% in cryopreservation in the solidified material of the present invention, while cell survival decreased in cryopreservation using the sample solution of Comparative Example 1. The rate had fallen to about 25%. Furthermore, in the cryopreservation in the solidified body of the present invention, a high cell viability was still maintained even after 12 months of cryopreservation, and the decrease in cell viability was only less than 15%.

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

Furthermore, when cells stored for a long period of time at -80°C are thawed and then stored as is at 4°C, when the solidified material of the present invention is used, the cell survival rate after storing for one day at 4°C is lower than that immediately before storage. While the cell survival rate was close to 100%, in Comparative Example 1 it was reduced to about 60%. Cells cryopreserved in the solidified material of the present invention still had a cell survival rate of 40% or more even after storage at 4°C for one week. This is because, during cryopreservation in the solidified material of the present invention, the cells that survived after thawing were cells that proceeded to proliferate normally, and that the matrix in the solidified material of the present invention during storage was This indicates that no damage was caused to the cells, thereby confirming that the matrix in the solidified body of the present invention has an extremely excellent cell preservation effect.

<Test Example 4: Evaluation of cell properties after cryopreservation in a solidified body using a test sample solution of primary human mesenchymal stem cells>
Cultured primary human mesenchymal stem cells (Lonza PT2501) were suspended in the test sample solutions (serum-free) of Example 1 and Comparative Example 1 at a concentration of 1×10 ⁶ cells/mL. Thereafter, the cell suspension containing each test sample solution was frozen in a -80°C freezer in a slow cell freezer (Nalgene® Mr. Frosty) to obtain a solidified body. The solidified material was stored at -80°C for 6 months. After 6 months, each solidified body was rapidly thawed in a 37°C hot bath, washed with αMEM, and then seeded at 2.5 × 10 ⁴ cells in a 6-well plate. After 4 days, HGF and IL in the culture medium were inoculated. -10 concentration was determined. For the quantification of HGF and IL-10, dedicated kits (Quantikine (registered trademark) ELISA Human HGF, catalog number DHG00, manufactured by R&D) and Quantikine (registered trademark) ELISA Human IL-10, catalog number D100B, manufactured by R&D were used, respectively. (manufactured by S.A., Inc.) according to the order of the procedure manual attached to the kit. The results are shown in Figures 5A and 5B.

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

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

Since the solidified material of the present invention does not contain cytotoxic compounds such as DMSO or ethylene glycol, cryopreservation in the solidified material of the present invention exposes biological samples to stress conditions, unlike conventional cryopreservation solutions. It has the remarkable effect of being able to be cryopreserved while maintaining its properties without causing any damage.

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

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

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

This result shows that the cryopreservation in the solidified body of the present invention is suitably applicable to the cryopreservation of stem cells, which is important to be cryopreserved in an undifferentiated state.

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

From the cell viability results shown in Table 1 together with the results of Test Example 1, the water-soluble polymer in the present invention is not limited to hyaluronic acid, and even other water-soluble polymers can be used. It can be seen that if the molecular aqueous solution has a predetermined endothermic peak temperature, it exhibits a high cell protection effect. In addition, from the results of Comparative Example 9 and Examples 3 and 4 (Comparative Example 9 with Production Example 1 added), it was found that by using a water-soluble polymer in combination with a saccharide or its salt having a smaller molecular weight, It was found that the solidified matrix showed good cytoprotective effect. As shown in the results of Test Example 2, by adding a low-molecular sugar or its salt to an aqueous polymer solution, the endothermic peak temperature of the sample solution was reduced to 0.7°C, the endothermic peak temperature of pure water. It is presumed that this was due to the adjustment being made so that they were close to each other. Comparative Example 10 is a test sample in which the low molecular weight saccharide of Production Example 2 (hyaluronic acid fragment sample with a viscosity average molecular weight of 2000) was added to a polymer aqueous solution of hyaluronic acid with a viscosity average molecular weight of 15000 (Comparative Example 5). Although it is a solution, as shown in the results of Test Example 2, the endothermic peak temperature of the DSC curve is -1.6 °C, lower than the endothermic peak temperature of Comparative Example 5 (-1.4 °C). As a result, the endothermic peak appearance temperature of pure water has moved away from 0.7°C. As a result, a cell survival rate as high as in the example was not obtained. These results show that higher cell survival rates can be obtained by adjusting the endothermic peak temperature of the test sample solution to a desired range by mixing low molecular weight saccharides or their salts into the aqueous polymer solution. Ta.

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

Furthermore, from Table 1, it can be seen that it is more preferable for the intrinsic viscosity (η) of the test sample solution to be in the range of about 0.20 dL/g or more and 0.95 dL/g or less in order to increase the cell survival rate. . When the intrinsic viscosity (η) is within the above predetermined range, the matrix containing the polymer solution helps expel water molecules from within the biological sample, and efficiently transports the expelled water molecules around the biological sample. It is believed that the saccharide can be replaced with a high-molecular and/or low-molecular saccharide. Furthermore, Table 1 shows that when the endothermic peak temperature of the DSC curve is −1.4° C. or lower and higher than 1.1° C., the cell survival rate after cryopreservation decreases. In other words, the fact that the endothermic peak temperature exceeds -1.4°C and is approximately 1.1°C or less is considered to indicate that the polymer solution can retain water molecules within the polymer matrix during freezing. It will be done.

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

<Test Example 7: Evaluation of cells in solidified body (evaluation of intracellular vitrification state)>
Cultured primary canine mesenchymal stem cells (cyagen C160) were suspended in the test sample solutions (serum-free) of Example 1 and Comparative Example 1 at a concentration of 1×10 ⁶ cells/mL. As a control, a control suspension in which cells were suspended in the test sample solution of Comparative Example 2 consisting of αMEM medium containing no cryopreservative was similarly prepared. Thereafter, 5 μL of the cell suspension containing each test sample solution and the control suspension were added to a hard glass sample plate (16φ x 0.12 mm), and a hard glass cover glass (12φ x 0.12 mm) was added. The temperature was lowered to −80° C. at a rate of 5° C./min using a cooling stage for microscopes manufactured by LinKam (THMS600), and an image of the solidified material was taken at −80° C. using a transmitted light microscope (Olympus BX53). The results are shown in Figures 7A-C.

FIG. 7A shows the results for the control suspension, and it can be seen that when using only the medium, the cells turned dark because ice crystals were generated inside the cells in the solidified body and light was diffusely reflected. FIG. 7B shows the results of Comparative Example 1 in which DMSO was the test sample. In FIG. 7B as well, the cells have turned dark and it can be seen that micro ice crystals have been formed. FIG. 7C shows the results of the solidified product of Example 1. It can be seen that the cells have turned bright, and the inside of the cells in the solidified body has been vitrified to an amorphous state.

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

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

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

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

When ice crystals form during freezing, visible light is blocked and brightness decreases. Therefore, it is preferable that the Munsell brightness is 2 or more in the solvent matrix region and the biological sample intracellular region (intracellular region). The fact that the Munsell lightness is at this level indicates that no ice crystals are formed in the solvent matrix region and the intracellular region (intracellular region), that is, the region is well vitrified. In Example 1, the Munsell brightness of the solvent region was 4, and the Munsell value of the intracellular region was 2, indicating that no ice crystals were formed in either region.

Further, the difference in brightness between the Munsell value in the solvent region and the Munsell value in the intracellular region was 2 in Examples 2 to 5, as in Example 1. In the solidified product of the present invention, the solvent region in the solidified product is in a state where ice crystals are not formed by adjusting the water-soluble polymer as described above. Therefore, this result that there is almost no difference between the brightness on the solvent matrix side and the brightness inside the biological sample means that the frozen state is almost the same in the matrix region and the intracellular region. That is, it is shown that in the solidified body of the present invention, ice crystals are not formed even in the region within the biological sample.

Furthermore, by comparing Examples 1 and 2 with Comparative Example 5, in which no hyaluronic acid with a viscosity average molecular weight of 1000 or less was added, and Comparative Example 10, in which hyaluronic acid with a viscosity average molecular weight of 2000 or less was added, By comparing Examples 3 and 4 with Comparative Example 9 in which hyaluronic acid with a viscosity average molecular weight of 1000 or less was not added, it was possible to adjust the difference between the brightness of the solvent matrix region and the brightness of the biological sample region. It has been found that this can be achieved by adding a saccharide or a salt thereof having a lower molecular weight than the water-soluble polymer to an aqueous polymer solution containing the water-soluble polymer or its salt. Table 1 shows that there is a good correlation between the brightness difference and the cell survival rate. It is desirable that the difference in brightness between the intracellular region and the solvent region is 3 or less. Furthermore, it is desirable that the brightness value of the solvent region is greater than or equal to the brightness value of the intracellular region. Therefore, in the solidified product of the present invention, the difference between the brightness of the solvent matrix region and the brightness of the biological sample region is adjusted to 3 or less by adding low molecular weight sugar or its salt. A good vitrification state is achieved in both the inner region and the solvent region.

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

Cells frozen in the solidified bodies of Examples 1-5 exhibited cell shrinkage rates of 38, 33, 33, 33, and 40%, respectively. In the cryopreservation in the solidified bodies of Examples 1 to 5, the discharge of water from the frozen cells is promoted, and the cells are well dehydrated during freezing. In the freezing of biological samples in Examples 1 to 5, in each case, water-soluble polymers and/or low molecular weight saccharides were present sufficiently close to the periphery of the cells, and there was a gap between the intracellular fluid and the polymer solution. The water molecules that move from inside the cell to the outside of the cell due to the osmotic pressure difference generated in The concentration of water in the cell supercools the water inside the cell, and the vapor pressure difference between the water and the supercooled water further promotes the movement of water molecules to the outside of the cell through the cell membrane, causing freezing and ice crystals within the cell. Preventing or suppressing the growth of Furthermore, since the cells are shrinking, it can be seen that water-soluble polymers and the like have not invaded the cells.

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

In cryopreservation in the solidified body of the present invention, damage to cells during freezing is significantly reduced due to good dehydration within cells during freezing, and as a result, thawed cells exhibit a high survival rate. it is conceivable that.

<Test Example 10: Evaluation of cryoprotection for various cells>
Cultured mouse-derived macrophage-like cell line (RAW264), human colon cancer-derived cells (Caco-2), and primary human lung microvascular endothelial cells (HMVEC) were each cultured at a concentration of 1×10 ⁶ cells/mL in Example 1 and Comparative Example 1 (serum-free). Thereafter, the cell suspension containing each test sample solution was frozen in a -80°C freezer in a slow cell freezer (Nalgene® Mr. Frosty) to obtain a solidified body. After storing the solidified bodies for 7 days, each solidified body was taken out and rapidly thawed in a hot 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 in the solidified material of the present invention, a high cell survival rate almost equal to or higher than that of Comparative Example 1 in which DMSO was used as a test sample was obtained for all cells. Ta. From this result, it was confirmed that various types of cells can be cryopreserved with a high cell survival rate in the solidified material of the present invention, regardless of whether they are primary cells or established cells, or regardless of the cell origin. Ta.

<Test Example 11: Evaluation of the effect of low molecular weight saccharides on cell viability>
Cultured primary canine mesenchymal stem cells (cyagen C160) were suspended in each test sample solution (serum-free) of Production Examples 1 to 3 at a concentration of 1×10 ⁶ cells/mL. Thereafter, the cell suspension containing each test sample solution was frozen in a -80°C freezer in a slow cell freezer (Nalgene® Mr. Frosty) to obtain a solidified body. After storing the solidified bodies for 7 days, each solidified body was taken out and rapidly thawed in a hot 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 intrinsic viscosity (η) of low molecular weight saccharides or their salts (viscosity average molecular weights of 1000, 2000, and 3000, respectively) that are smaller than water-soluble polymers is 0. 08, 0.14, and 0.19, which are outside the desired range of 0.2 dL/g or more and 0.95 dL/g or less. Test Example 11 revealed that such low molecular weight saccharides or their salts alone cannot provide cell survival effects. In Production Example 3, a cell survival rate of about 10% was observed, which is presumed to be due to hyaluronic acid having a molecular weight larger than 3000 that may be contained in the test sample of Production Example 3.

<Test Example 12: HPLC analysis of subcritically treated hyaluronic acid sample>
A 1 wt % aqueous solution of the test samples of Production Examples 1 to 3 was prepared, filtered through a 0.45 μm membrane filter (manufactured by Millipore), and the components of each test sample were analyzed by HPLC. As the mobile phase, A solution: 16mM NaH ₂ PO ₄ aqueous solution, B solution: 800mM NaH ₂ PO ₄ aqueous solution were used, and ZORBAX NH ₂ (manufactured by Agilent Technologies, Co., Ltd., column size φ4.6 x 250 mm, particle size 5 μm) was used. The components were separated using a normal phase HPLC column at a flow rate of 1.0 mL/min, a column temperature of 40° C., and a detection wavelength of 210 nm. The gradient conditions were as follows: mobile phase B concentration: 0% (0 minutes) → mobile phase B concentration: 100% (60 minutes). The results are shown in Figures 11A-C. In addition, as standard samples, HA02, a disaccharide, HA04, a tetrasaccharide, HA06, a hexasaccharide, HA08, an octasaccharide, and HA10, a decasaccharide (all manufactured by Izuron) were prepared at a concentration of 0.2 wt%. Aqueous solutions containing each oligosaccharide of hyaluronic acid were prepared and subjected to HPLC analysis in the same manner. The results are shown in FIG. 11D.

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

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

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

From the above results, in the solidified body of the present invention, the inside of the biological sample is stably vitrified in the same way as the matrix part, and this prevents or suppresses the growth of ice crystals within the biological sample during freezing. I understand that. The solidified material of the present invention basically does not require the addition of cell-permeable and cytotoxic compounds such as DMSO or ethylene glycol, and/or serum or serum-derived proteins, and has a high cell survival rate. It can be seen that this method has the remarkable effect of being able to freeze and preserve biological samples. In the solidified body of the present invention, cells are well protected and their properties are maintained.

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

Claims (13)

A solidified body comprising a matrix and a biological sample containing an aqueous solvent and a water-soluble polymer or a salt thereof having an intrinsic viscosity of 0.2 to 0.95 dL/g in the aqueous solvent ,
The biological sample has an orthogonal projected area that is smaller than 1 and reduced to 1/3.5 or more compared to before solidification,
When visible light is transmitted, the difference between the brightness in the Munsell color system of the area occupied by the biological sample and the brightness in the Munsell color system of the area occupied by the matrix is 2 or less, when viewed from the light exit side,
In the DSC curve of the heating process of the matrix obtained under the measurement conditions (1) to (2) below using a differential scanning calorimeter (DSC), no endothermic peak is confirmed, or an endothermic peak is confirmed. A solidified material having a temperature of more than -1.4°C and less than 1.1°C.
(DSC measurement conditions)
(1) After holding at 20°C for 1 minute, lower the temperature to -80°C at a cooling rate of 5°C/min.
(2) After holding at -80°C for 1 minute, raise the temperature to 20°C at a temperature increase rate of 10°C/min. The solidified product according to claim 1, which is a solid frozen product. The solidified material according to claim 1 or 2, which contains the water-soluble polymer or its salt in a content of 0.1 to 20 w/v% or less. In the DSC curve of the temperature raising process obtained under the measurement conditions of (1) and (2) above, the endothermic amount of the endothermic peak in the temperature increasing process is 0 J/g, or the corresponding endothermic amount of the reference solution made of water. The solidified material according to any one of claims 1 to 3, which has a content of 65% or less. The solidified body according to any one of claims 1 to 4, wherein the biological sample is a cell, and the cell is a mammalian cell. The solidified body according to claim 5, wherein the biological sample is a cell, and the cell is a mammalian mesenchymal stem cell, a mammalian blood cell, or a mammalian endothelial cell. The solidified material according to any one of claims 1 to 6, which is used as a solution for biological sample administration after being melted. The amount of HGF produced by the biological sample contained in the biological sample administration solution is the amount of HGF produced after the biological sample in the solidified body containing DMSO instead of the water-soluble polymer or its salt is thawed. The solidified body according to claim 7, which is comparatively suppressed. The amount of IL-10 produced by the biological sample contained in the biological sample administration solution is the IL-10 produced after the biological sample in the solidified body containing DMSO instead of the water-soluble polymer or its salt is thawed. 8. The solidified material according to claim 7, wherein the production amount is increased compared to that of the solidified material. A method for producing a solidified body containing a biological sample, the method comprising:
When a water-soluble polymer or a salt thereof having an intrinsic viscosity of 0.2 to 0.95 dL/g in an aqueous solvent is dissolved in the aqueous solvent and frozen at -80°C, the biological sample solidifies. It has an orthogonal projected area that is smaller than 1 and reduced to 1/3.5 or more compared to before,
When frozen at -80°C, when visible light is transmitted, the brightness in the Munsell color system of the area occupied by the biological sample and the Munsell value of the area other than the area occupied by the biological sample, as seen from the light exit side. The difference in brightness in the color system is 2 or less,
In the DSC curve of the heating process obtained using a differential scanning calorimeter (DSC) under the measurement conditions (1) and (2) below, no endothermic peak is observed, or the temperature at which the endothermic peak is observed is -1. .Preparing an aqueous polymer solution adjusted to have a temperature exceeding 4°C and below 1.1°C;
a step of including the biological sample in the polymer aqueous solution;
A method for producing a solidified body, comprising the steps of cooling and freezing the aqueous polymer solution containing the biological sample.
(DSC measurement conditions)
(1) After holding at 20°C for 1 minute, lower the temperature to -80°C at a cooling rate of 5°C/min.
(2) After holding at -80°C for 1 minute, raise the temperature to 20°C at a temperature increase rate of 10°C/min. The solidification according to claim 10, wherein the step of cooling and freezing the aqueous polymer solution containing the biological sample is performed by cooling and freezing to a temperature of -27°C or less by a slow freezing method at a cooling rate of 10°C/min or less. How to manufacture the body. The method for producing a solidified body according to claim 10 or 11, wherein the solidified body contains the water-soluble polymer in a content of 0.1 to 20 w/v% or less. In the DSC curve of the temperature raising process obtained under the measurement conditions of (1) and (2) above, the endothermic amount of the endothermic peak in the temperature increasing process is 0 J/g, or the corresponding endothermic amount of the reference solution made of water. The method for producing a solidified body according to any one of claims 10 to 12, wherein the solidified body has a content of 65% or less.

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