JP2023091093A - Cryopreservation liquid - Google Patents

Abstract

To provide cryopreservation liquids for biological samples with high cell viability, to provide cryopreservation methods for biological samples, and to provide methods for stably storing biological samples for a long period of time.SOLUTION: Provided are a cryopreservation liquid for biological samples, which has a viscosity average molecular weight of more than 3000 and 500000 or less in a solvent, and contains a polysaccharide containing a six-membered ring structure in its main chain or a salt thereof; a cryopreservation method comprising including a biological sample in a cryopreservation liquid containing a polysaccharide or a salt thereof; a method for storing a biological sample; and a cryopreservation agent for biological samples consisting of a polysaccharide or a salt thereof.SELECTED DRAWING: None

Description

The present invention relates to cryopreservation solutions. The present invention also relates to a biological sample cryopreservation method using a cryopreservation solution, a biological sample preservation method, and a biological sample cryopreservation agent.

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

In the cryopreservation mechanism of cells, if ice crystals grow inside cells during the freezing and/or thawing process, cell membranes and intracellular structures are damaged, and cell proteins are denatured, resulting in fatal cell damage. It is known to receive Therefore, during cryopreservation of cells, it is important to prevent intracellular freezing, and low-molecular-weight compounds such as methyl sulfoxide (DMSO), glycerin, and propylene glycol are usually used for intracellular permeation for cryopreservation of cells. It has been used as a cryoprotective reagent of the type by adding it to a buffer solution such as a culture medium (Patent Document 1). Among these, DMSO is the most commonly used and has a good effect of protecting cells and cell organelles. However, in intracellular permeation type cryopreservation solutions, low-molecular-weight compounds, which are cryoprotective reagents, permeate into cells, so there is concern about the effect of cryoprotective reagents on cells (Non-Patent Document 1).

Therefore, attempts have been made to use natural cryoprotectants as cryoprotective reagents instead of chemical substances. For example, disaccharides, oligosaccharides, or polysaccharides are known to be added to buffers, such as culture media, as non-permeating cryoprotective agents.

Also, a method of retaining biological components in a crosslinked body forming a hydrogel is being studied. In Patent Document 2, a raw material hyaluronic acid having a weight average molecular weight of 5,000 to 4,000,000 is modified with a side chain having a substituent that reacts with a hydroxyl group to form a crosslinked structure. Preservatives for ingredients are described. Modified hyaluronic acid reacts with the hydroxyl groups of a compound having multiple hydroxyl groups such as polyvinyl alcohol to form a crosslinked product obtained by crosslinking the modified hyaluronic acid. is used as The molecular weight of the hydrogel described in Patent Document 2 as an actual preservative is presumed to be several million or more. In Patent Document 2, the biological components are stored in a refrigerator at about 4° C., and the storage period is about several days. In addition, Patent Document 3 discloses a fructan having a five-membered ring structure as an active ingredient of a cell preservation solution.

JP-A-63-216476 WO2016/076317 JP 2012-235728 A

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

Intracellularly penetrating cryoprotectants promote dehydration of cells, thereby slowing down the rate of intracellular ice crystal formation and inhibiting ice crystal formation. In particular, DMSO easily penetrates into cells, and is therefore effective for cryopreservation of cells with complex structures such as mammalian cells. chemicals are cytotoxic. It is thought that as the intracellular penetration of the cryoprotectant progresses and the 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 been reported to affect differentiation of ES cells (Cryobiology. 2006 Oct;53(2):194-205.). Therefore, the use of DMSO as a cryoprotective reagent may not be suitable for cell preservation when maintenance of undifferentiated or functional stem cells is required. In addition, when samples are stored for a long time using DMSO, it is essential to store the samples in liquid nitrogen or in an atmosphere that requires handling management. be done.

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

With the biological component preservative described in Patent Document 2, as described above, the tested preservation period was only a few days, and a better cryopreservation technique is required for preservation on a scale of 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 further, to dissolve the gel for recovery of biological components from the hydrogel after storage. The use of additives is also required. However, if such an additive is introduced into the cryopreservation method, it can be used for research tests, but it is difficult to use it for actual medical use due to the risk of contamination with impurities.

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

A method using a non-permeating cryoprotective agent is also known, but such a preservation agent alone has little effect on cells, but cannot provide a sufficient preservation effect. Therefore, a combination of an intracellular penetrating type compound such as intracellular DMSO, glycerin, and propylene glycol and a non-permeating type substance is commercially available in which the amount of the compound is reduced or replaced. However, there have been no reports of a practical cryoprotectant that exhibits a favorable cryopreservation effect and is composed solely of natural ingredients such as biological constituents.

The present invention has been made in view of the above problems, and provides a cryopreservation solution for appropriately preserving a biological sample. In particular, chemical substances such as dimethyl sulfoxide (DMSO), propylene glycol (PG), and ethylene glycol (EG) are basically not used, and biological samples are prepared without basically adding serum or serum-derived proteins. It is an object of the present invention to provide a cryopreservation solution and a cryopreservation method, a cryopreservation agent for biological samples, and a method for stably preserving biological samples.

The present invention relates to a cryopreservation solution for biological samples containing a polysaccharide or a salt thereof having a viscosity average molecular weight of more than 3000 and not more than 500000 and having a six-membered ring structure in the main chain in a solvent. Polysaccharide salts are desirably metal salts, halogen salts or sulfates. As metal salts, salts of alkali metals or alkaline earth metals are desirable. Sodium, potassium, calcium and the like are selected as alkali metals or alkaline earth metals. Chlorine, bromine, and the like can be used as halogen. The polysaccharide or salt thereof having a viscosity average molecular weight of more than 3,000 and not more than 500,000 and containing a six-membered ring structure in the main chain is desirably contained as a main component in the cryopreservation solution. As used herein, the term "main component" refers to the component with the highest weight ratio among the components dissolved in the solvent. Components other than the main component are subcomponents.

In the present invention, the polysaccharide containing a six-membered ring structure in the main chain or a salt thereof preferably has a viscosity-average molecular weight of 400,000 or less, particularly 200,000 or less. This is because the viscosity can be adjusted to be low and it is easy to handle as a cryopreservation solution.

Moreover, in the present invention, dextran is desirably excluded from polysaccharides as the main component. In addition, the cell cryopreservation medium of the present invention may contain dextran as an auxiliary component such as a cell protective agent, which is not a main component.

Cryopreservation solutions for biological samples containing polysaccharides containing pentoses, hexoses or uronic acids or combinations thereof as repeating units are preferred.

Cryopreservation solutions for biological samples containing polysaccharides containing amino sugars as repeating units are preferred.

It is desirable that the functional groups of the polysaccharide or its salt used in the cryopreservation medium for biological samples of the present invention are not modified.

Moreover, a sulfated polysaccharide can be used as the polysaccharide contained in the cryopreservation solution for biological samples.

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

A cryopreservation solution for biological samples comprising a polysaccharide that is hyaluronic acid or pullulan is preferred.

A cryopreservation solution for biological samples is preferred, wherein the concentration of the polysaccharide or salt thereof in the cryopreservation solution is 5 w/v% or more and 20 w/v% or less.

A cryopreservation solution for biological samples, which is an intracellular impermeable cryopreservation solution, is preferred.

A cryopreservation solution for a biological sample is preferred, wherein the biological sample is a cell, tissue, or tissue-like substance that is a membrane or an aggregate.

A cryopreservation solution for a biological sample, wherein the biological sample is mesenchymal stem cells, blood cells, endothelial cells, or tissue for transplantation, is preferred.

A cryopreservation solution for a biological sample, wherein the biological sample is sperm, ovum, or fertilized egg, is preferred.

The present invention also provides a method for freezing a biological sample, wherein the cryopreservation solution of the present invention is used for cooling and freezing by slow freezing at a cooling rate of preferably 10° C./min or less, preferably 1° C./min or less. Regarding.

The present invention also includes a biological sample in a cryopreservation solution containing, in a solvent, a polysaccharide or a salt thereof having a viscosity-average molecular weight of more than 3,000 and not more than 500,000 and containing a six-membered ring structure in the main chain. subjecting the cryopreservation solution containing the biological sample to freezing; and preserving the cryopreservation solution containing the biological sample at a temperature of −27° C. or less. related to the cryopreservation method. Polysaccharide salts are desirably metal salts, halogen salts or sulfates. As metal salts, salts of alkali metals or alkaline earth metals are desirable. Sodium, potassium, calcium and the like are selected as alkali metals or alkaline earth metals. Chlorine, bromine, and the like can be used as halogen.

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

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

In the biological sample cryopreservation method of the present invention, the polysaccharide in the cryopreservation solution is desirably a polysaccharide other than dextran.

Preferably, the biological sample cryopreservation method includes the step of including the biological sample in the cryopreservation solution before cooling the biological sample.

Preferably, the biological sample is a cell, tissue, or a tissue-like substance that is a membrane or an aggregate.

A method of cryopreserving a biological sample, wherein the biological sample is sperm, ovum, or fertilized egg, is preferred.

The present invention also provides a method for preserving a biological sample, wherein the biological sample has a viscosity average molecular weight of more than 3000 and not more than 500000, and a polysaccharide or a salt thereof containing a six-membered ring structure in its main chain. and stored at −27° C. or lower, and the biological sample immediately before storage when the biological sample is stored for a period of at least 5 months in the presence of the polysaccharide or its salt It relates to a method characterized in that preservation exhibits less than a 5% loss of viability based on the viability of the sample. Polysaccharide salts are desirably metal salts, halogen salts or sulfates. As metal salts, salts of alkali metals or alkaline earth metals are desirable. Sodium, potassium, calcium and the like are selected as alkali metals or alkaline earth metals. Chlorine, bromine, and the like can be used as halogen.

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

The storage reduces the viability of the biological sample by less than 10% relative to the viability of the biological sample immediately prior to storage when the biological sample is stored in the presence of the polysaccharide or salt thereof for a period of at least 6 months. A method of preserving a biological sample, characterized in that the preservation exhibits

When the biological sample is stored at 4° C. for 1 day after being frozen at -27° C. or lower and then thawed, the survival rate is reduced by less than 5% based on the survival rate of the biological sample immediately after thawing. A method of preserving a biological sample is preferred, characterized by:

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

A method of preserving a biological sample in which the biological sample is a cell is preferred.

A method of preserving a biological sample, wherein the biological sample is mammalian cells, is preferred.

Preferably, the biological sample is mammalian mesenchymal stem cells, mammalian blood cells, or mammalian endothelial cells.

The present invention also provides a six-membered ring structure in its main chain having a viscosity average molecular weight of more than 3000 and not more than 500000 for storing biological samples, preferably in vitro, at a temperature of -27°C or less. It relates to the use of a polysaccharide or a salt thereof having Polysaccharide salts are desirably metal salts, halogen salts or sulfates. As metal salts, salts of alkali metals or alkaline earth metals are desirable. Sodium, potassium, calcium and the like are selected as alkali metals or alkaline earth metals. Chlorine, bromine, and the like can be used as halogen.

In the present invention, the polysaccharide containing a six-membered ring structure in the main chain or a salt thereof preferably has a viscosity-average molecular weight of 400,000 or less, particularly 200,000 or less. This is because the viscosity can be adjusted to be low and it is easy to handle as a cryopreservation solution.

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.

In the present invention, the polysaccharide used for preserving the biological sample is desirably a polysaccharide other than dextran.

The present invention also relates to the use of a polysaccharide or a salt thereof having a viscosity average molecular weight of more than 3000 and less than or equal to 500000 and containing a six-membered ring structure in the main chain for cryopreserving biological samples. Polysaccharide salts are desirably metal salts, halogen salts or sulfates. As metal salts, salts of alkali metals or alkaline earth metals are desirable. Sodium, potassium, calcium and the like are selected as alkali metals or alkaline earth metals. Chlorine, bromine, and the like can be used as halogen.

In the present invention, the polysaccharide containing a six-membered ring structure in the main chain or a salt thereof preferably has a viscosity-average molecular weight of 400,000 or less, particularly 200,000 or less. This is because the viscosity can be adjusted to be low and it is easy to handle as a cryopreservation solution.

In the present invention, it is desirable that the polysaccharide used for cryopreserving a biological sample is a polysaccharide other than dextran.

The present invention also relates to a cryopreservation agent for biological samples, comprising a polysaccharide or a salt thereof having a viscosity-average molecular weight of more than 3,000 and not more than 500,000 and having a six-membered ring structure in its main chain. Polysaccharide salts are desirably metal salts, halogen salts or sulfates. As metal salts, salts of alkali metals or alkaline earth metals are desirable. Sodium, potassium, calcium and the like are selected as alkali metals or alkaline earth metals. Chlorine, bromine, and the like can be used as halogen.

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

It is desirable that the polysaccharide as the main component of the cryopreservation agent is a polysaccharide other than dextran.

Cryopreservation agents for biological samples comprising polysaccharides containing pentoses, hexoses or uronic acids or combinations thereof as repeating units are preferred.

Cryopreservation agents for biological samples containing polysaccharides containing amino sugars as repeating units are preferred.

The functional groups of the polysaccharide or its salt used in the biological sample cryopreservation agent of the present invention are desirably unmodified.

A biological sample cryopreservation agent containing a polysaccharide having a viscosity average molecular weight of 10,000 or more or a salt thereof is preferred.

A biological sample cryopreservation agent comprising a polysaccharide that is hyaluronic acid or pullulan is preferred.

According to the present invention, it is possible to appropriately preserve biological samples such as cells and tissues without using a cell-permeable and toxic compound such as DMSO or ethylene glycol, which is an intracellular cryoprotectant. can. Furthermore, since the cryopreservation solution of the present invention is intracellular impermeable, it exhibits a high cryopreservation effect, but has low toxicity to cells.

Also, since it does not contain serum and/or serum-derived proteins, it is not contaminated with bacteria or viruses. It is possible to add proteins that are not contaminated with bacteria or viruses. In addition, even a cell-permeable and toxic compound can be used at a low concentration that does not impair cell functions.

The "viscosity average molecular weight" of the polysaccharide used in the present invention is obtained by the following method and calculation formula

ここで、η_sp：多糖類の還元粘度［ｍＬ／ｇ］、η_r：多糖類の相対粘度［－］、Ｃ：多糖類の濃度［ｇ／ｍＬ］である。
（６）多糖類の濃度と、多糖類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（多糖類濃度＝０）の値を極限粘度とする。 Intrinsic viscosity measurement:
(1) A predetermined amount of NaCl is dissolved in deionized water at 30° C. to prepare a 0.2 M NaCl solution (standard solution).
(2) A polysaccharide sample is dissolved in a standard solution at 30°C to prepare a stock solution.
The viscosity of each of the standard solution and the stock solution is measured and adjusted so that the relative viscosity of the stock solution to the standard solution is 2.0 to 2.4.
(3) Dilute the stock solution at 30°C with the standard solution at 30°C to 5/4, 5/3 and 5/2 times.
(4) Measure the viscosities 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 the stock solution and the diluted solution by the viscosity of the standard solution, and the reduced viscosity is derived based on the following formula.

Here, η _sp : reduced viscosity of polysaccharide [mL/g], η _r : relative viscosity of polysaccharide [−], C: concentration of polysaccharide [g/mL].
(6) Plot the relationship between the polysaccharide concentration and the reduced viscosity of the polysaccharide, and draw an approximate straight line. The value of the intercept (polysaccharide concentration=0) of the approximate straight line is defined as the intrinsic viscosity.

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。 Viscosity average molecular weight:
The viscosity average molecular weight is calculated from the intrinsic viscosity.

The viscosity-average molecular weight M is obtained from the intrinsic viscosity derived by measurement in the Mark Hoing-Sakurada formula described above, and the values of K and α disclosed in literatures and the like.

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 "Handbook of Polymer Materials" (edited by The Society of Polymer Science, Japan), and are not published. Calculations of the viscosity average molecular weight can be made using the values shown.

For example, K=3.6×10 ⁻⁴ and α=0.78 for hyaluronic acid, and K=9×10 ⁻⁴ and α=0.5 for pullulan and gelatin according to literature values.

In addition, it is desirable to use an aqueous solvent such as water as the solvent used to prepare the cryopreservation solution of the present invention. In particular, it is preferably an isotonic solution in which the salt concentration, sugar concentration, etc. are adjusted with sodium ions, potassium ions, calcium ions, etc. so that the osmotic pressure is approximately the same as that of body fluids or cell fluids. Specifically, for example, water, physiological saline, phosphate buffered saline (PBS), which is a physiological saline with a buffering effect, Dulbecco's phosphate buffered saline, Tris buffered saline ( Tris Buffered Saline; TBS), HEPES buffered saline, etc., balanced salt solutions such as Hank's balanced salt solution, Ringer's solution, lactated Ringer's solution, acetated Ringer's solution, bicarbonate Ringer's solution, or D-MEM, E-MEM, αMEM, RPMI- Animal cell culture basal media such as 1640 medium, Ham's F-12, Ham's F-10 and M-199, and other commercially available media can be used.

The cryopreservation solution for biological samples of the present invention contains, as a cryoprotectant, a polysaccharide or a salt thereof having a viscosity-average molecular weight of more than 3,000 and not more than 500,000, and having a six-membered ring structure in its main chain. A portion can be frozen into an amorphous glass. As a result, cell rupture due to ice crystal formation is suppressed, and since there is no volumetric expansion of water, damage to cells and cell membranes can be suppressed. Even if the viscosity-average molecular weight of the polysaccharide is 3000 or less or exceeds 500000, the survival rate of the biological sample after freezing becomes low. Also, the polysaccharide must contain a six-membered ring structure in its main chain. This is because polysaccharides having a six-membered ring structure in the main chain easily form a polymer network structure around cells through hydrogen bonding, and it is thought that vitrification of the solvent portion can be achieved even at low concentrations. . Therefore, even if the polysaccharide concentration is low, a sufficient cell cryopreservation effect can be obtained. On the other hand, in the case of polysaccharides having a five-membered ring structure, such as fructans, it is difficult to stably form such a polymer network structure, and as a result, the polysaccharide concentration must be increased. It is estimated to be.

In the present invention, the polysaccharide containing a six-membered ring structure in the main chain or a salt thereof preferably has a viscosity-average molecular weight of 400,000 or less, particularly 200,000 or less. This is because the viscosity can be adjusted to be low and it is easy to handle as a cryopreservation solution.

In addition, in the present invention, it is desirable not to contain dextran as a polysaccharide.

Furthermore, since polysaccharides are biological constituents, in addition to low toxicity, the cryopreservation solution for biological samples containing the polysaccharide of the present invention should be an intracellular non-penetrating cryopreservation solution. Nevertheless, since it exhibits a high cryopreservation effect that could not be obtained with conventional techniques, it is possible to remarkably reduce damage to the biological sample and changes in the properties of the biological sample.

In addition, since the cryopreservation solution for biological samples of the present invention does not contain intracellular permeable chemical substances such as DMSO and ethylene glycol, cytotoxicity and cell properties reported for DMSO do not occur. It is considered that there is no risk of adverse effects of Therefore, the properties of the biological sample can be maintained during and after cryopreservation. It is possible to add cytotoxic chemical substances such as DMSO and ethylene glycol at low concentrations that do not impair cell functions.

Moreover, since it does not contain serum or serum-derived proteins, it is not contaminated with bacteria or viruses. It is possible to add proteins that are not contaminated with bacteria or viruses.

Further, since the cryopreservation solution for a biological sample of the present invention has a glass transition point around −23° C.±4, the method of freezing a biological sample, the method of cryopreserving a biological sample, and the method of preserving a biological sample of the present invention. According to the article, without using cytotoxic chemicals such as DMSO or proteins such as serum, biological samples such as cells and tissues are appropriately stabilized for a long period of time at a temperature of -27 ° C or less, that is, in a deep freezer. can be saved. It is possible to add proteins that are not contaminated with bacteria or viruses. It is also possible to add cytotoxic chemicals such as DMSO and ethylene glycol 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. 2 is a diagram showing cell viability of primary human mesenchymal stem cells in cryopreservation using the test cryopreservation medium of the present invention. FIG. 4 is a diagram showing analysis results of a cryopreservation solution by differential scanning calorimetry. FIG. 3 is an enlarged view showing analysis results of a cryopreservation solution by differential scanning calorimetry. FIG. 4 is a diagram showing analysis results of a cryopreservation solution containing ultrapure water by differential scanning calorimetry. FIG. 3 is a diagram showing analysis results of a cryopreservation solution containing DMSO by differential scanning calorimetry. 1 is a diagram showing analysis results of a cryopreservation solution containing a test sample of Example 1 by differential scanning calorimetry. FIG. FIG. 2 is a diagram showing the effect of long-term preservation of primary human mesenchymal stem cells in cryopreservation using the test cryopreservation solution of the present invention. FIG. 2 is a diagram showing the effect of long-term storage of primary human mesenchymal stem cells in cold storage using the test cryopreservation medium of the present invention. FIG. 2 shows the amount of HGF produced in primary human mesenchymal stem cells after cryopreservation using the test cryopreservation medium of the present invention. FIG. 2 is a diagram showing the production amount of IL-10 in primary human mesenchymal stem cells after cryopreservation using the test cryopreservation medium of the present invention. FIG. 2 shows the expression levels of undifferentiated biomarkers in primary human mesenchymal stem cells after cryopreservation using the test cryopreservation medium of the present invention. FIG. 2 shows the effects of cryopreservation solutions containing various polysaccharides of the present invention on primary canine mesenchymal stem cells. It is a figure which shows the addition effect of various fragments. FIG. 3 shows the intracellular vitrification state after freezing in a control study. FIG. 2 is a diagram showing intracellular vitrification state after freezing using the test cryopreservation medium of Comparative Example 1. FIG. 1 is a diagram showing intracellular vitrification using the test cryopreservation medium of Example 1. FIG. FIG. 4 is a diagram showing the difference in brightness in intracellular vitrified states. FIG. 2 is a diagram showing the brightness difference between the solvent and the cell by quantifying the brightness difference in the intracellular vitrified state according to the Munsell brightness (0 to 10). FIG. 3 is a diagram showing the cell area of frozen primary canine mesenchymal stem cells in a cryopreservation solution. FIG. 2 is a diagram showing cryoprotective effects on various cells during cryopreservation using the test cryopreservation solution of the present invention. FIG. 3 shows the cell viability of primary canine mesenchymal stem cells in cryopreservation using the saccharide of the present invention. FIG. 3 shows HPLC analysis results of test samples according to various production examples. FIG. 3 shows HPLC analysis results of test samples according to various production examples. FIG. 3 shows HPLC analysis results of test samples according to various production examples. FIG. 2 shows HPLC analysis results for a standard curve of saccharides having a given number of saccharides. FIG. 4 shows the HPLC analysis results of test samples having polymer chains of the present invention. FIG. 10 shows cell viability with test samples without polysaccharide structures. FIG. 2 is a diagram showing cell viability when fructans were used as test samples.

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

In the present invention, the polysaccharide containing a six-membered ring structure in the main chain or a salt thereof preferably has a viscosity-average molecular weight of 400,000 or less, particularly 200,000 or less. This is because the viscosity can be adjusted to be low and it is easy to handle as a cryopreservation solution.

The polysaccharide is not particularly limited as long as it has a polysaccharide skeleton in the main chain and contains a six-membered ring structure in the main chain. Included are those bound by a bond and derivatives thereof. For example, monosaccharides include monosaccharides having 3 to 7 carbon atoms, or monosaccharides in which hydroxyl groups and/or hydroxymethyl groups of monosaccharides are substituted, for example, hydroxyl groups and/or hydroxymethyl groups are substituted with carboxyl groups. , an amino group, an N-acetylamino group, a sulfooxy group, a methoxycarbonyl group and a carboxymethyl group. However, in the present invention, dextran is desirably excluded from polysaccharides.

Monosaccharides having 3 to 7 carbon atoms include triose, tetrose, pentose, hexose and heptose, among which pentose and hexose are preferred. Pentose includes ribose, arabinose, xylose, lyxose, xylulose, ribulose, deoxyribose and the like. Hexoses include glucose, mannose, galactose, fructose, sorbose, tagatose, fucose, fucrose, rhamnose and the like.

Substituted monosaccharides include monosaccharides in which one or more hydroxymethyl groups of the monosaccharide are substituted with carboxyl groups, methoxycarbonyl groups or carboxymethyl groups, one or more hydroxyl groups of the monosaccharides are substituted with amino groups or N A monosaccharide substituted with an acetylamino group, a monosaccharide substituted with a sulfooxy group for one or more hydroxyl groups of the monosaccharide, and the like. Also, substituted monosaccharides are substituted monosaccharides in which more than one of the hydroxymethyl and hydroxyl groups of the monosaccharide have been substituted with functional groups or combinations thereof as described above. good too.

For example, uronic acid etc. can be illustrated as a monosaccharide substituted with the carboxyl group. Uronic acids include, for example, glucuronic acid, iduronic acid, mannuronic acid and galacturonic acid. An amino sugar etc. can be illustrated as a monosaccharide substituted with the amino group. Amino sugars include, for example, glucosamine, galactosamine, mannosamine and muramic acid. Monosaccharides substituted with N-acetylamino groups include, for example, N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine and N-acetylmuramic acid. Galactose-3-sulfate and the like can be exemplified as monosaccharides substituted with a sulfooxy group. Monosaccharides having multiple substituents include, for example, N-acetylglucosamine-4-sulfate, iduronic acid-2-sulfate, glucuronic acid-2-sulfate, N-acetylgalactosamine-4-sulfate, neuraminic acid and N - Acetylneuraminic acid and the like.

Polysaccharides used in the present invention are, for example, polymers containing monosaccharides as repeating units as described above. The polysaccharide used in the present invention may further have its chemical structure partially modified or substituted as appropriate to the extent that it does not gel. For example, polysaccharides used in the present invention may be sulfated polysaccharides, such as chondroitin sulfate, in which one or more hydroxyl groups have been replaced with sulfooxy groups. However, it is most desirable that the polysaccharide used in the present invention has no functional group modification, that is, no substituent is introduced into the sugar chain. Functional groups of polysaccharides, especially hydrophilic groups such as OH, _NH , and COOH groups, are presumed to contribute to the protection of biological samples and the vitrification of solvents, so these functional groups have been modified. This is because the lack thereof is advantageous for improving the survival rate of the biological sample.

Also, the polysaccharide salt used in the present invention may be a metal salt, halogen salt or sulfate of the polysaccharide. As metal salts, salts of alkali metals or alkaline earth metals are desirable. Sodium, potassium, calcium and the like are selected as alkali metals or alkaline earth metals. Chlorine, bromine, and the like can be used as halogen. Polysaccharide salts are believed to lower the freezing point of the solvent, thereby contributing to the vitrification of the solvent.

Polysaccharides used in the present invention can be polysaccharides containing, for example, pentoses, hexoses or uronic acids or combinations thereof as repeating units. Polysaccharides may also be polysaccharides containing amino sugars as repeating units. For example, polysaccharides include modified sugars of hyaluronic acid, pullulan, chondroitin sulfate, methylcellulose, or hydroxyethyl starch. Preferably, the polysaccharide may be hyaluronic acid or pullulan.

In addition, in the present invention, it is desirable to use a polysaccharide other than dextran as the polysaccharide.

Polysaccharides may be of natural origin or may be chemically synthesized. Commercially available polysaccharides may be used as they are. Monosaccharides forming the polysaccharide backbone may also be naturally occurring monosaccharides, modified or substituted naturally occurring monosaccharides, or chemically synthesized monosaccharides. For example, preferably the monosaccharides that form the polysaccharide backbone are biological constituents. However, monosaccharides are not limited to this. Monosaccharides forming the polysaccharide skeleton preferably contain hydrophilic groups such as hydroxyl groups and carboxyl groups. In particular, the polysaccharide used in the present invention preferably has equatorial hydroxyl groups in its structure. It is believed that this allows the solvent water to be better trapped in the matrix formed by the polymer chains when frozen.

The cryopreservation solution containing the polysaccharide of the present invention desirably does not contain dimethylsulfoxide (DMSO). In addition, it is desirable that the cryopreservation solution containing the polysaccharide of the present invention does not contain a cell-penetrating cytotoxic compound such as ethylene glycol. In addition, in the cryopreservation solution containing the polysaccharide of the present invention, the action of the high-molecular sugar chain of the polysaccharide restricts the molecular movement of water in the solvent, so that the water is solidified in a vitrified state without crystallizing and/or Or can be frozen. The freezing method called the normal vitrification method is a method that is applied to cells that have a particularly low survival rate after thawing. In this method, the salt concentration in the remaining solution increases due to the elimination, and an osmotic pressure difference occurs between the inside and outside of the cell, dehydrating the inside of the cell and vitrifying the inside of the cell. Therefore, such methods involve increasing the concentration of the solute (cryoprotectant) and increasing the cooling rate to facilitate vitrification of the water. However, it has been known that increasing the osmotic pressure difference causes greater damage to cells, and that recrystallization occurs during dissolution, resulting in damage to cells. Moreover, it is accompanied by technical difficulty.

Moreover, since the cryopreservation solution containing the polysaccharide of the present invention does not contain serum or serum-derived proteins, it is not contaminated with bacteria or viruses. It is possible to add proteins that are not contaminated with bacteria or viruses.

According to the cryopreservation solution containing the polysaccharide or salt thereof of the present invention, solvent molecules can be trapped in the matrix formed by the polymer chains during the cooling process. This dehydrates and vitrifies the inside of the cell, suppressing the formation of ice crystals inside the cell, and also weakens the osmotic shock in the cell during freezing, which is a problem with conventional vitrification methods. can. Since the cryoprotectant is a polysaccharide, the cytotoxicity is low in the first place, and there is no need to increase the solute concentration to limit the molecular movement of water. is considered low. Moreover, unlike the conventional vitrification method, the cryopreservation solution of the present invention does not require a high cooling rate for reducing osmotic shock during cooling. Therefore, according to the cryopreservation solution of the present invention, a biological sample can be efficiently cryopreserved by a simple method with reduced toxicity.

As a polysaccharide or a salt thereof as a cryopreservative contained in the cryopreservation solution of the present invention, it is preferable to use those having a viscosity-average molecular weight of more than 3000 and about 500000 or less. In the present invention, it is desirable that the viscosity-average molecular weight of the polysaccharide or its salt containing a six-membered ring structure in the main chain is 400,000 or less, particularly 200,000 or less. This is because the viscosity can be adjusted to be low and it is easy to handle as a cryopreservation solution.

In addition, the "viscosity average molecular weight" of the polysaccharide used in the present invention means a value calculated from the following method and formula.

ここで、η_sp：多糖類の還元粘度［ｍＬ／ｇ］、η_r：多糖類の相対粘度［－］、Ｃ：多糖類濃度［ｇ／ｍＬ］である。
（６）多糖類濃度と多糖類の還元粘度との関係をプロットし、近似直線を引く。近似直線の切片（多糖類濃度＝０）の値を極限粘度とする。
粘度平均分子量：
粘度平均分子量は、極限粘度から算出する。

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。ヒアルロン酸の場合は、Ｋ＝３．６×１０^-4およびα＝０．７８を代入して、粘度平均分子量Ｍを求める。実施例で用いられているプルランおよびゼラチンには、文献値よりＫ＝９×１０^-4、α＝０．５を、また、デキストランには、Ｋ＝６．３×１０^-8、α＝１．４、フルクタンには、Ｋ＝１．６×１０^-3、α＝０．４５を用いる。 A method for measuring the intrinsic viscosity and a method for calculating the viscosity-average molecular weight using the intrinsic viscosity are described below.
Intrinsic viscosity measurement:
(1) A predetermined amount of NaCl is dissolved in deionized water at 30° C. to prepare a 0.2 M NaCl solution (standard solution).
(2) A polysaccharide sample is dissolved in a standard solution at 30°C to prepare a stock solution. If the polysaccharide sample is obtained in solution, the solid content after removing the solvent from the solution is taken as the polysaccharide sample. In the case of a mixed sample containing multiple polysaccharides, each polysaccharide is separated and fractionated, and then the solvent is removed from each substance to obtain a sample of each water-soluble polymer. If the polysaccharide is unknown, the substance is identified by HPLC, LC-MS, LC-IR, or the like. In addition, if multiple unknown polysaccharides are contained, each component is separated and fractionated, and the substance is identified by HPLC, LC-MS, LC-IR, etc. for each polysaccharide, and the viscosity average molecular weight is determined as described later. calculate. Even if the polysaccharide is mixed with impurities, if the viscosity is not affected (for example, impurities such as metal salts), the mixture is used as the polysaccharide sample. In addition, if impurities that affect the calculation of the viscosity-average molecular weight are included, the measurement is performed after removing the impurities or fractionating the polysaccharides. At this time, the viscosity of each of the standard solution and the stock solution is measured, and the relative viscosity of the stock solution to the standard solution is adjusted to 2.0 to 2.4.
(3) Dilute the stock solution at 30°C with the standard solution at 30°C to 5/4, 5/3 and 5/2 times.
(4) Measure the viscosities 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 the stock solution and the diluted solution by the viscosity of the standard solution, and the reduced viscosity is derived based on the following formula.

Here, η _sp : reduced viscosity of polysaccharide [mL/g], η _r : relative viscosity of polysaccharide [−], C: concentration of polysaccharide [g/mL].
(6) Plot the relationship between the polysaccharide concentration and the reduced viscosity of the polysaccharide, and draw an approximate straight line. The value of the intercept (polysaccharide concentration=0) of the approximate straight line is defined as the intrinsic viscosity.
Viscosity average molecular weight:
The viscosity average molecular weight is calculated from the intrinsic viscosity.

The viscosity-average molecular weight M is obtained from the intrinsic viscosity derived by measurement in the Mark Hoing-Sakurada formula described above, and the values of K and α disclosed in literatures and the like. For hyaluronic acid, K=3.6×10 ⁻⁴ and α=0.78 are substituted to determine the viscosity average molecular weight M. For pullulan and gelatin used in the examples, K=9×10 ⁻⁴ and α=0.5 from literature values, and for dextran, K=6.3×10 ⁻⁸ and α=1. .4, using K=1.6×10 ⁻³ and α=0.45 for fructans.

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 Material Handbook" (edited by The Society of Polymer Science, Japan), and are not published. Calculation of the viscosity average molecular weight is performed using the values provided.

If the polysaccharide to be used is unknown as described above, K and α are determined after identifying the polysaccharide by HPLC, LC-MS, LC-IR, or the like.

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

By setting the viscosity-average molecular weight of the polysaccharide or its salt within the above range, the cryopreservation solution of the present invention containing the polysaccharide or its salt in the solvent is amorphous in the frozen state when cooled. The glassy state is stabilized. Cells are stably and efficiently cryopreserved because cooling and freezing do not cause cell damage. Therefore, the survival rate of cells in the biological sample after thawing the biological sample after cryopreservation is high. If the viscosity-average molecular weight of the polysaccharide is 3000 or less, vitrification is difficult to occur well. On the other hand, if the viscosity average molecular weight of the polysaccharide is more than 500,000, the viscosity will be significantly increased, the solubility will be lowered, and the prepared solution will foam, resulting in poor handling. Therefore, when using a naturally-derived high-molecular-weight polysaccharide or a commercially available high-molecular-weight sugar chain, it is preferable to perform treatments such as hydrolysis, enzymatic treatment, and subcritical treatment to adjust the molecular weight. The viscosity-average molecular weight is particularly preferably 10,000 or more. Polysaccharides having a viscosity-average molecular weight of 400,000 or less are preferable, 200,000 or less are more preferable, and 150,000 or less are particularly preferable. This is because the viscosity can be adjusted to be low and it is easy to handle as a cryopreservation solution.

The cryopreservation solution of the present invention has a viscosity average molecular weight of more than 3000 and about 500000 or less, and by using a polysaccharide or a salt thereof containing a six-membered ring structure in its main chain, human- or bovine-derived serum or It exhibits a high cytoprotective effect without requiring the addition of protein components such as serum-derived components (eg albumin). Therefore, there is no risk of infectious diseases, and it is thought that biologics will not be affected by lot-to-lot differences. In addition, it is possible to add a protein that does not cause infection. In addition, since the cryopreservation medium of the present invention has low cytotoxicity, cells after thawing exhibit a high viability. According to the results of thermal analysis of the cryopreservation solution of the present invention by differential scanning calorimetry (DSC), the cryopreservation solution of the present invention has a glass transition temperature around -23°C ± 4°C. Furthermore, the cryopreservation solution of the present invention not only suppresses the formation of ice crystals in the cooling process, but also suppresses the recrystallization of water during the subsequent heating process, that is, during thawing. In other words, it is considered that the cryopreservation using the polysaccharide of the present invention stabilizes the vitreous state of the biological sample in the frozen state.

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

As described above, the cryopreservation method using the cryopreservation solution of the present invention does not require a large cooling rate for cell protection during cooling. The cryopreservation solution of the present invention is cooled to −27° C. or below, which is the glass transition temperature of the cryopreservation solution of the present invention. Therefore, cells and tissues to be preserved can be stably frozen and preserved. Since the low temperature of −150° C. required for cryopreservation of ordinary cells is not necessarily required, a special container for cryopreservation in liquid nitrogen or the preparation of liquid nitrogen can be eliminated. For this reason, operations related to cryopreservation of biological samples can be significantly simplified. In addition, since the cryopreservation solution of the present invention can also suppress recrystallization of water during thawing, the use of the cryopreservation solution of the present invention enables a series of steps of freezing, preserving, and thawing a biological sample. can be performed easily and efficiently without requiring special techniques. Of course, it is also possible to cryopreserve a biological sample using liquid nitrogen using the cryopreservation solution of the present invention.

Furthermore, the cryopreservation solution of the present invention is considered to have low cytotoxicity because it is a non-permeating cryoprotective reagent. In addition, since the cryopreservation agent of the cryopreservation solution of the present invention is a polysaccharide, it is believed that it does not change the properties of cells. Therefore, it is considered that a biological sample can be cryopreserved while maintaining cell characteristics.

The cryopreservation solution of the present invention contains the polysaccharide of the present invention or a salt thereof at a concentration of approximately 1 w/v% or more and 50 w/v% or less. If the concentration is lower than 5 w/v%, the solvent portion may not be vitrified satisfactorily. On the other hand, if the concentration is higher than 20 w/v%, the viscosity may become too high and the handleability may deteriorate. For example, the polysaccharide or its salt concentration is preferably 5 w/v% or more, particularly preferably 10 w/v% or more. Moreover, the concentration of the polysaccharide or its salt is preferably 50 w/v% or less, particularly preferably 20 w/v% or less. Preferably, the concentration of the polysaccharide or its salt is 5 w/v% or more and 20 w/v% or less.

The cryopreservation solution of the present invention may further contain a cytoprotective component. Such cytoprotective ingredients include, for example, compounds having a viscosity average molecular weight greater than 3000 and less than polysaccharides of 500,000 or less. When such compounds are present in cryopreservation solutions, water molecules near cell membranes can be replaced by cytoprotective components to inhibit ice crystal formation and growth near cell membranes, thereby further inhibiting cell membrane damage.

Specifically, for example, the cytoprotective component is a saccharide that is a constituent of the polysaccharide of the present invention or a fragment of the polysaccharide of the present invention. For example, monosaccharides, disaccharides or oligosaccharides having a viscosity-average molecular weight of less than 3000, preferably 2000 or less, more preferably 1000 or less are exemplified. In the case of monosaccharides, disaccharides, and compounds considered to be single molecules, the molecular weight is clearly specified from the structural formula, so in the present invention, the molecular weight specified from the structural formula is assumed to be the viscosity average molecular weight. deal.

The cytoprotective component that can be used in the present invention is not particularly limited as long as it does not impair the effects of the present invention, but particularly preferred examples include cleavage products (fragments) of polysaccharides such as glycosaminoglycans. can. A glycosaminoglycan cleavage product can be a monosaccharide, a disaccharide thereof, or an oligosaccharide thereof making up the glycosaminoglycan.

Preferably, the glycosaminoglycan cleavage product is a hyaluronic acid cleavage product. Preferably, therefore, the glycosaminoglycan cleavage product is glucuronic acid or N-acetylglucosamine, or a di- or oligosaccharide consisting thereof. More preferably, it may be glucuronic acid or its disaccharide or oligosaccharide.

The “cleavage product” of the present invention means a polysaccharide having a viscosity average molecular weight of 3000, which is considered to be obtained when a polysaccharide is subjected to hydrolysis, enzymatic treatment, subcritical treatment, or the like. and less than 500,000 polysaccharides with a viscosity average molecular weight. Thus, the cleavage products can be monosaccharides and/or polymers of varying degrees of polymerization of monosaccharides and/or mixtures thereof that are constituents of the corresponding polysaccharides. As used herein, the term “subcritical treatment” refers to contacting a subcritical fluid as an extraction solvent that has been brought into a subcritical state under conditions of a predetermined temperature and pressure, and a raw material to be extracted, that is, a high-molecular-weight polysaccharide. means to let For example, water exhibits neither a liquid nor a gas when the pressure is raised to 22.12 MPa or higher and the temperature is raised to 374.15° C. or higher. This point is called the critical point of water, and hot water at a temperature and pressure below the critical point is called subcritical water. A desired component can be obtained from the raw material to be extracted using the hydrolysis action of this subcritical water. Conditions for subcritical treatment of high-molecular-weight polysaccharides are, for example, a temperature of 150° C. or higher and 350° C. or lower, and a subcritical treatment pressure of at least the saturated vapor pressure at each temperature. It can be 0.5 MPa or more and 25 MPa or less. After the subcritical treatment, components with a predetermined molecular weight or less can be separated and recovered and used as cleavage products in the present invention. Moreover, hydrolysis and enzymatic treatment are not particularly limited, and commonly used reagents and treatment methods can be used without any problem.

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

Since the cryopreservation solution of the present invention is a non-permeating cryoprotective reagent, the biological sample to be cryopreserved using the cryopreservation solution of the present invention is not particularly limited. It can be used for cryopreservation of various types of cells. Also, the origin of the cells is not particularly limited. Since the cryopreservation solution of the present invention can effectively suppress ice crystal formation and recrystallization during freezing and thawing, it can be used favorably for mammalian cells and the like having complex structures. Therefore, it can be applied to cryopreservation of cells of various animal species such as mice, dogs, and humans. Furthermore, the cryopreservation solution of the present invention is known to have greater damage during freezing than common cells for culture, and the so-called conventional slow freezing method inevitably causes a significant decrease in viability. It is particularly suitable for cryopreservation of reproductive cells such as stem cells, early embryos, eggs, sperm, and fertilized eggs. In addition, since the cryopreservation solution of the present invention does not contain a cell-permeable and cytotoxic chemical substance such as DMSO that can act as a differentiation factor, it can be used to preserve cells that need to be maintained undifferentiated. For example, stem cells for regenerative medicine applications can also be cryopreserved without the risk of differentiation. It is possible to add chemicals such as DMSO and ethylene glycol at low concentrations where the above risks are not a problem.

In addition, since no serum or serum-derived protein is added, there is no contamination with bacteria or viruses. In addition, it is possible to add a protein that does not cause infection.

In addition, since the cryopreservation solution of the present invention is excellent in the effect of stabilizing the vitreous state in the frozen state, it is known to be difficult to preserve cells such as ova with large sizes and fertilized eggs. , and cryopreservation of organized cell structures, tissues and tissue-like objects, such as tissues obtained in regenerative medicine.

That is, the cryopreservation solution of the present invention can achieve a high viability when used for a biological sample selected from cells, tissues, or tissue-like substances such as membranes or aggregates. In particular, the cryopreservation solution of the present invention, regardless of primary cells or established cells, mesenchymal stem cells; hematopoietic stem cells; neural stem cells; somatic stem cells such as bone marrow stem cells and germline stem cells; Cryopreservation such as cryopreservation, in particular, cryopreservation of primate stem cells, which are said to be less tolerant to freezing than mice, cryopreservation of tissues for transplantation, and cryopreservation of germ cells in reproductive medicine.

In addition, by using a polysaccharide that is a biological component as the polysaccharide used in the present invention, it is possible to thaw a cryopreserved biological sample and use it as it is as a solution for cell administration. Furthermore, in the case of a tissue or tissue-like substance, it is also possible to add a polysaccharide at the stage of organization and to cryopreserve it as it is. This is believed to alleviate post-implantation problems. In such cases, a preferred polysaccharide for cryopreservation solutions of the invention is hyaluronic acid. In particular, hyaluronic acid having a viscosity-average molecular weight of more than 3,000, preferably more than 5,000, and less than or equal to 60,000, more preferably less than or equal to 20,000.

The cryopreservation solution of the present invention is a cryopreservation solution for slow freezing. That is, in the freezing method using the cryopreservation solution of the present invention, the cooling rate is preferably 10° C./min or less, more preferably about 1° C./min or less. At this cooling rate, it is considered that the inside of the cells is appropriately dehydrated, vitrification of the intracellular fluid occurs well, and a high cell cryoprotective effect is obtained.

In the cryopreservation method of the present invention, a biological sample is placed in a cryopreservation solution containing, in a solvent, a polysaccharide having a six-membered ring structure in its main chain or a salt thereof, having a viscosity average molecular weight of more than 3000 and not more than 500000. freezing a cryopreservation solution containing the biological sample; and preserving the cryopreservation solution containing the biological sample at a temperature of −27° C. or lower. The polysaccharide salt may be a metal salt, halogen salt or sulfate salt of the polysaccharide. As metal salts, salts of alkali metals or alkaline earth metals are desirable. Sodium, potassium, calcium and the like are selected as alkali metals or alkaline earth metals. Chlorine, bromine, and the like can be used as halogen. In a deep freezer at -80 ° C., a cryopreservation solution containing a biological sample can be frozen at a cooling rate of 10 ° C./min or less, preferably at a cooling rate of about 1 ° C./min. Rapid operation for rapid cooling required in the vitrification process is not required. Therefore, it is considered that the operability is improved and at the same time a stable preservation effect can be obtained.

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

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.

As a solvent for the cryopreservation solution of the present invention, it is desirable to use an aqueous solvent such as water. In particular, it is preferably an isotonic solution in which the salt concentration, sugar concentration, etc. are adjusted with sodium ions, potassium ions, calcium ions, etc. so that the osmotic pressure is approximately the same as that of body fluids or cell fluids. Specifically, for example, water, physiological saline, phosphate buffered saline (PBS), which is a physiological saline with a buffering effect, Dulbecco's phosphate buffered saline, Tris buffered saline ( Tris Buffered Saline (TBS), HEPES buffered saline, etc., balanced salt solutions such as Hank's balanced salt solution, Ringer's solution, lactated Ringer's solution, acetated Ringer's solution, bicarbonate Ringer's solution, etc., but are not limited thereto. In addition, the solvent may contain other optional ingredients 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% glucose aqueous solution, or the like. A cell culture medium may also be used as a solvent for the cryopreservation solution of the present invention. The culture medium is not particularly limited, and examples thereof include commercially available medium, D-MEM, E-MEM, αMEM, RPMI-1640 medium, Ham's F-12, Ham's F-10, M-199, etc. basal medium for animal cell culture, general culture medium for various cells or tissues. Therefore, the cryopreservation medium of the present invention may be added to the culture medium or cell suspension after cell culture so that the cryopreservation agent has a desired concentration.

Moreover, the cryopreservation solution of the present invention may be pH-adjusted as necessary. For example, when a polysaccharide having a carboxylic acid group or the like is used as the polysaccharide of the present invention, the cryopreservation solution may exhibit acidity. In such cases, it is thought that by adjusting the pH of the cryopreservation solution to a neutral solution, the solution can be made more suitable for the survival of the cells to be cryopreserved, and the survival rate of the cells will be improved. Salts used for pH adjustment are not limited, and those commonly used for pH adjustment of aqueous solutions can be used.

In the cryopreservation method for a biological sample using the cryopreservation solution of the present invention, the vitrified state is stabilized and the toxicity is low, so the biological sample can be stably preserved for a long period of time. As used herein, long-term stable storage means, for example, that the viability of a biological sample, such as cells, after thawing using the cryopreservation solution of the present invention is based on the viability of cells immediately before storage, After 5 months, less than about 10%, preferably less than 5%, or after 6 months, less than 20%, preferably less than 10%, or after 12 months, less than 15% It means that there is only a degree of reduction, preferably less than 30%. In the present specification, long-term stable storage means, for example, when cells are frozen and stored at −80° C. for a long period of time, then thawed, and then stored at 4° C., after thawing, 24 hours after thawing, there is less than a 5% decrease in viability relative to cell viability immediately after thawing. It is considered that the cryopreservation solution of the present invention allows the cells to be cryopreserved under less stressful conditions than the DMSO solution. Therefore, the cryopreservation solution of the present invention can provide a significantly higher cell viability after thawing compared to cryopreservation using a conventional cryopreservation solution such as a 10% DMSO solution. Furthermore, not only cells immediately after thawing, but also cells stored in cold storage after thawing can exhibit high cell viability. According to the cryopreservation solution of the present invention, cells can be stably cryopreserved for a long period of time without changing the properties of the cells.

EXAMPLES The present invention will be specifically described based on Examples, but the present invention is not limited to these. Also, hereinafter, hyaluronic acid manufactured by lifecore biomedical is sodium hyaluronate, but is described as "hyaluronic acid" for simplification.

Cryopreservation agent and cryopreservation solution <Preparation of test sample and sample solution>
・Example 1
Polymer hyaluronic acid having an average molecular weight of 1 million (manufactured by Shanghai Easier Industrial Development) and water were mixed at a ratio of 20:100 in a pressure vessel 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. After that, the subcritically processed product was dried by a freeze-drying method or a spray-drying method. As a result, a test sample of hyaluronic acid having a viscosity-average molecular weight of about 10,000, that is, the cryopreservation agent of the present invention was obtained. The intrinsic viscosity was 0.49 dL/g.

By dissolving 1 g of this test cryopreservation agent in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent is water), a test cryopreservation solution of Example 1 was obtained (that is, test freezing 10 w/v % concentration of hyaluronic acid sample in storage solution). In the examples, production examples, and comparative examples of the present invention, including this Example 1, the αMEM medium is used as the solvent, but ultrapure water may be used as the solvent instead of the αMEM medium. Further, in the DSC measurement described later, ultrapure water is used as the solvent instead of αMEM. In addition, each test sample obtained in the following examples is prepared as a cryopreservation agent of a cryopreservation solution so that it has a predetermined concentration using an appropriate solvent in each evaluation test described later. Used.

・Manufacturing example 1
A test sample of a hyaluronic acid fragment having a viscosity-average molecular weight of 1,000 was obtained in the same manner as in Example 1, except that the subcritical treatment was performed for 7 minutes. The intrinsic viscosity was 0.08 dL/g.

・Manufacturing example 2
A test sample of a hyaluronic acid fragment having a viscosity-average molecular weight of 2,000 was obtained in the same manner as in Example 1, except that the subcritical treatment was performed at a treatment time of 5 minutes. The intrinsic viscosity was 0.14 dL/g.

・Production example 3
A test sample of a hyaluronic acid fragment having a viscosity-average molecular weight of 3,000 was obtained by performing the same operation as in Example 1, except that the subcritical treatment was performed at a treatment time of 4 minutes. The intrinsic viscosity was 0.19 dL/g.

・Example 2
Hyaluronic acid with a viscosity-average molecular weight of 50000 (manufactured by lifecore biomedical; powder) was used as a test sample for the cryopreservation agent of the present invention. By dissolving 1 g of this test sample in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent is water) as a solvent, a test cryopreservation solution of Example 2 was obtained (that is, test Hyaluronic acid concentration of the test sample in the cryopreservation solution is 10 w/v%). The intrinsic viscosity was 1.67 dL/g.

・Example 3
1 g of hyaluronic acid (manufactured by lifecore biomedical; powder) with a viscosity-average molecular weight of 15000 was dissolved in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent is water), and the concentration of hyaluronic acid was 10 w/v%. To the test sample solution, the test sample of Production Example 1 (hyaluronic acid fragment sample with a viscosity average molecular weight of 1000) is added at a final concentration of 1 w / v%, and the test cryopreservation solution of Example 3 is prepared. Obtained. The intrinsic viscosity was 0.65 dL/g.

・Example 4
The pH of the test cryopreservation solution of Example 3 (containing hyaluronic acid (manufactured by lifecore biomedical) with a viscosity average molecular weight of 15000 and the test sample of Production Example 1) is adjusted to neutral using 10 mM Tris-HCl. Thus, the test cryopreservation solution of Example 4 was obtained. The intrinsic viscosity was 0.65 dL/g.

・Example 5
By dissolving 1 g of pullulan (manufactured by Tokyo Chemical Industry Co., Ltd.) having a viscosity average molecular weight of 373,000 in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent is water), the test of Example 5 was performed. A cryopreservation solution for use was obtained (that is, the test sample in the cryopreservation solution for testing had a pullulan concentration of 10 w/v%). The intrinsic viscosity was 0.55 dL/g.

・Example 6
The test sample of Production Example 1 (hyaluronic acid fragment sample with a viscosity average molecular weight of 1000) was added to the test cryopreservation solution of Example 5 (including pullulan with a viscosity average molecular weight of 373000) at a final concentration of 1 w / v%. The cryopreservation liquid for the test of Example 6 was obtained by addition. The intrinsic viscosity was 0.55 dL/g.

・Example 7
The test of Example 7 was carried out by adjusting the pH of the test cryopreservation solution of Example 6 (containing pullulan with a viscosity average molecular weight of 373000 and the test sample of Production Example 1) to neutral using 10 mM Tris-HCl. It was used as a cryopreservation solution for

・Example 8
The test sample of Production Example 1 (hyaluronic acid fragment sample with a viscosity average molecular weight of 1000) was added to the test cryopreservation solution of Example 5 (including pullulan with a viscosity average molecular weight of 373000) at a final concentration of 5 w / v%. It was added to prepare a cryopreservation solution for testing. The intrinsic viscosity was 0.55 dL/g.

・Example 9
By adjusting the pH of the test cryopreservation solution of Example 8 (containing pullulan with a viscosity average molecular weight of 373000 and the test sample of Production Example 1) to neutral using 10 mM Tris-HCl, It was used as a cryopreservation solution for testing. The intrinsic viscosity was 0.55 dL/g.

・Example 10
1 g of hyaluronic acid (manufactured by lifecore biomedical; powder) with a viscosity-average molecular weight of 15000 was dissolved in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent is water), and the concentration of hyaluronic acid was 10 w/v%. Sucrose (manufactured by Nacalai Tesque Co., Ltd.) was added to the test sample solution in Example 10 at a final concentration of 1 w / v% to prepare a test cryopreservation solution. The intrinsic viscosity was 0.65 dL/g.

・Example 11
1 g of hyaluronic acid (manufactured by lifecore biomedical; powder) with a viscosity-average molecular weight of 15000 was dissolved in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent is water), and the concentration of hyaluronic acid was 10 w/v%. Glucuronic acid (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was added to the test sample solution in Example 11 at a final concentration of 1 w/v% to prepare a test cryopreservation solution. The intrinsic viscosity was 0.65 dL/g.

・Comparative example 1
DMSO (manufactured by Nacalai Tesque Co., Ltd., cell culture grade) was used as a test sample. By dissolving 1 mL of this test sample in 10 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent is water) as a solvent, a test cryopreservation solution of Comparative Example 1 was obtained (that is, test DMSO concentration of test sample in cryopreservation solution is 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
By dissolving 1 g of gelatin (viscosity average molecular weight of 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, freezing for testing in Comparative Example 3 was performed. A stock solution was obtained (ie, 10 w/v % gelatin concentration of the test sample in the test cryopreservation medium). The intrinsic viscosity was 0.50 dL/g.

・Comparative example 4
By dissolving 1 g of high molecular weight hyaluronic acid (manufactured by Shanghai Easier Industrial Development Co., Ltd.) having a viscosity average molecular weight of 1000000 in 100 mL of αMEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent is water) as a solvent, Comparative Example 4 A test cryopreservation solution was obtained (that is, the concentration of hyaluronic acid, which is a test sample in the test cryopreservation solution, is 1 w / v%). The test cryopreservation solution of Comparative Example 4 was prepared to have a hyaluronic acid concentration of 1 w/v% because a concentration of 10 w/v% in the medium could not be obtained due to handling problems. The intrinsic viscosity was 17.2 dL/g.

・Experimental example 1
By dissolving 1 g of dextran having a viscosity average molecular weight of 42500 (manufactured by MP; powder) in 10 mL of αMEM medium as a solvent, a test sample solution of Experimental Example 1 was obtained (i.e., a test sample in a test sample solution dextran concentration of 10 w/v%). The intrinsic viscosity was 0.19 dL/g.

・Comparative example 6
A fructan derived from shallots is obtained according to Example 2 of JP-A-2012-235728.

About 1 kg of dried scallion as a raw material is pulverized, mixed with water for extraction in which 15 g of calcium hydroxide is suspended, extracted at room temperature for 1 hour, and then heated and extracted at 95 ° C. for 1 hour, After that, it was heated and extracted at 60° C. overnight. Activated carbon was added to the extract for deodorization, and then filtered through diatomaceous earth to remove residue and activated carbon.

The resulting extract was passed through an ultrafiltration membrane to purify fructans.

The obtained purified liquid was heat sterilized once, and then decolorized again by adding activated charcoal. This purified liquid was sterilized by heating at 85° C. for 1 hour, freeze-dried and pulverized to obtain 400 g of purified fructan powder.

The viscosity-average molecular weight of the obtained purified fructan powder was measured and found to be 12,000. The intrinsic viscosity was 0.11 dL/g.

By dissolving 1 g of this fructan powder in 10 mL of αMEM medium as a solvent, a test sample solution of Comparative Example 6 was obtained (that is, the fructan concentration of the test sample in the test sample solution was 10 w/v%).

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

Cell suspensions containing each test cryopreservative were then frozen in a -80°C freezer in a slow cell freezer (Nalgene® Mr. Frosty). The frozen cell suspension containing each test sample was stored at -80°C for 7 days and then rapidly thawed in a 37°C hot bath. The cell suspension containing each test cryopreservation medium after thawing was evaluated for cell viability by trypan blue staining immediately after thawing. The results are shown in FIG.

FIG. 1 shows cells cryopreserved in medium without cryopreservation medium, i.e., in the test cryopreservation medium of Comparative Example 2, or in the test cryopreservation medium of Examples 1 and 2 and Comparative Examples 1 and 4. , cell viability after thawing. From FIG. 1, it can be seen that the test cryopreservation solutions of Examples 1 and 2 exhibited good preservation effects. In particular, in the case of the test cryopreservation solution of Example 1, in which the hyaluronic acid sample having a viscosity-average molecular weight of about 10,000 obtained by subcritical treatment of hyaluronic acid is the cryopreservation agent in the cryopreservation solution, it exceeds 90%. A high preservation effect was obtained. This preservative effect was higher than that of Comparative Example 1 containing DMSO, which is a commonly used cryopreservative, as a cryopreservative. It can be seen that the cryopreservation solution of the present invention is an excellent cryopreservation solution with low cytotoxicity. In Comparative Example 4, in which high-molecular-weight hyaluronic acid with a high viscosity-average molecular weight of 1,000,000 was used as the test sample, almost no cell-protecting effect was observed. In addition, in Comparative Example 2 containing no cryopreservative, the cell viability was almost 0%. From these results, it was confirmed that polysaccharides having a viscosity-average molecular weight of about 200,000 or less are effective as cryoprotectants. In addition, since the cryopreservation agent of the present invention exhibits a high cell preservation effect even when prepared as a cryopreservation solution using a culture medium such as αMEM medium as a solvent, the cryopreservation agent of the present invention can be used as it is for cells. After suspending the cells in addition to the culture medium, they can be frozen and stored. It is considered possible to maintain higher cell viability and cell properties without the need to centrifuge the cells to be stored.

<Test Example 2: Evaluation of analysis of cryopreservation solution by differential scanning calorimetry>
The test samples of Examples 1 and 2 and Comparative Example 2 were adjusted to a final concentration of 10% using ultrapure water instead of the αMEM medium, and used as samples for differential scanning calorimetry. Each sample was scanned on a differential scanning calorimeter (DSC) as follows.
(1) After being held at 20°C for 1 minute, the temperature was lowered to -80°C at a free rate.
(2) After being held at -80°C for 1 minute, the temperature was raised to 20°C at a rate of 10°C/min.

Results are shown in Figures 2A and 2B. In Comparative Example 2 in which the test sample was ultrapure water (that is, the ultrapure water sample), only the ice crystal melting peak of free water was observed as shown in FIG. 2A. In Example 2, in which the test sample is hyaluronic acid having a viscosity-average molecular weight of 50,000, a shift in the ice melting peak and a depression of the freezing point were observed compared to Comparative Example 2, and a glass transition was confirmed at around -20°C. (Fig. 2B). In Example 1, the melting peak of ice crystals was extremely small and almost not observed, and further, as shown in FIG. 2B, a glass transition was confirmed around −23° C.±4° C.

Next, the test samples of Example 1 and Comparative Examples 1 and 2 were adjusted to a final concentration of 10% using ultrapure water instead of the αMEM medium, and used as samples for differential scanning calorimetry. Each sample was scanned on a differential scanning calorimeter (DSC) as follows.
(1) After being held at 20°C for 1 minute, the temperature was lowered to -80°C at a rate of 5°C/min.
(2) After being held at -80°C for 1 minute, the temperature was raised to 20°C at a rate of 10°C/min.

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

In Comparative Example 2, in which the test sample was ultrapure water, a very large exothermic peak accompanying ice crystal formation was observed during the temperature drop process, as shown in FIG. 3A. Also, during the heating process, a large amount of heat of fusion was observed as ice crystals melted. In Comparative Example 1, in which the test sample was DMSO, as shown in FIG. 3B, only small peaks associated with ice crystal formation were observed, and almost no peaks associated with ice crystal melting were observed. This indicates that the cryopreservation solution containing the test sample (DMSO) of Comparative Example 1 is close to a vitrified state in the frozen state. In the cryopreservation solution containing the test sample of Example 1 as a cryopreservation agent, as shown in FIG. much smaller than the peak. Therefore, in the cryopreservation solution containing the test sample of Example 1, almost no ice crystals are formed, and it is considered to be in a very good vitrification state.

<Test Example 3: Evaluation of long-term storage effect in cryopreservation using a test cryopreservation solution for primary human mesenchymal stem cells>
The cultured primary human mesenchymal stem cells (Lonza PT2501) were suspended in the test cryopreservation medium (serum-free) of Example 1 and Comparative Example 1 at a concentration of 1×10 ⁶ cells/mL. Cell suspensions containing each test cryopreservative were then frozen in a -80°C freezer in a slow cell freezer (Nalgene® Mr. Frosty). After storing the frozen cell suspension containing each test cryopreservation medium at -80 ° C. for up to 12 months, remove the cell suspension containing each test cryopreservation medium for a predetermined storage period, 37 It was quickly thawed in a warm bath at ℃. The cell viability of the cell suspension containing each test cryopreservation medium after thawing was evaluated by trypan blue staining immediately after thawing. The results are shown in FIG. 4A. Alternatively, cell suspensions frozen at -80°C for 3 months were thawed and then stored at 4°C for one day or one week. Cell viability of cell suspensions after storage at 4°C was assessed by trypan blue staining. The cell viability after storage at 4° C. was calculated assuming that the cell viability immediately after thawing (that is, immediately before storage) was 100%. The results are shown in Figure 4B.

From the results shown in FIG. 4A, it can be seen that the cryopreservation solution of the present invention exhibits a high cell viability of 95% or more, which is almost unchanged even after storage for 5 months. On the other hand, in the cryopreservation solution containing the test sample (DMSO) of Comparative Example 1, a decrease in cell viability was already observed after two months of cryopreservation. After 3 months, the cell survival rate in cryopreservation in Comparative Example 1 is less than 50%. This result was in contrast to the cryopreservation solution of the present invention, which still maintained high cell viability after 3 months. After 6 months of cryopreservation, the cryopreservation solution of the present invention shows less than 10% reduction in viability, while the cryopreservation solution of Comparative Example 1 shows a decrease in cell viability to about 25%. rice field. Furthermore, in cryopreservation with the cryopreservation solution of the present invention, a high cell viability was still maintained even after 12 months of cryopreservation, and the decrease in cell viability was only less than 15%.

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

In addition, when the cells stored at -80 ° C. for a long time are stored at 4 ° C. as they are after thawing, in the cryopreservation solution of the present invention, the cell viability after storage at 4 ° C. for 1 day is the cell viability immediately before storage. was nearly 100%, while in Comparative Example 1 it was reduced to about 60%. Even after one week of storage at 4°C, the cryopreservation solution of the present invention still showed a cell viability of 40% or more. This indicates that, in the cryopreservation solution of the present invention, the cells that survived after thawing were substantially growing normally, and that the cryopreservation solution did not damage the cells during storage. This shows that the cryopreservation solution of the present invention has a very excellent cell preserving effect.

<Test Example 4: Evaluation of properties after cryopreservation using a test sample of primary human mesenchymal stem cells>
The cultured primary human mesenchymal stem cells (Lonza PT2501) were suspended in the test cryopreservation medium (serum-free) of Example 1 and Comparative Example 1 at a concentration of 1×10 ⁶ cells/mL. Cell suspensions containing each test cryopreservative were then frozen in a -80°C freezer in a slow cell freezer (Nalgene® Mr. Frosty). The frozen cell suspension containing each test cryopreservation medium was cryopreserved at −80° C. for 6 months. Six months later, the cell suspension containing each test cryopreservation medium was rapidly thawed in a 37°C hot bath, washed with αMEM, and seeded in 6-well plates at 2.5 x 10 ⁴ cells each. Days later, HGF and IL-10 concentrations in the culture medium were quantified. For quantification of HGF and IL-10, dedicated kits (Quantikine (registered trademark) ELISA Human HGF, catalog number DHG00, manufactured by R&D), Quantikine (registered trademark) ELISA Human IL-10, catalog number D100B, R&D (manufactured by Co., Ltd.), and performed according to the order of the procedure manual attached to the kit. Results are shown in Figures 5A and 5B.

As shown in FIG. 5A, cryopreservation in the presence of the cryopreservation medium of Comparative Example 1 containing DMSO as a cryopreservation agent resulted in high HGF production in cells after thawing. On the other hand, in cryopreservation using the cryopreservation medium of the present invention containing hyaluronic acid with a viscosity average molecular weight of 10000 as a cryopreservation agent, the amount of HGF produced in the cells after thawing was low. was less than 1/3 of the Results in the presence of DMSO indicate that cells were stressed upon storage in the presence of DMSO. Since such high HGF production was not observed in cell preservation in the presence of the cryopreservation agent of the present invention, it can be seen that cells are stably protected in the cryopreservation medium of the present invention.

As shown in FIG. 5B, the amount of IL-10 production was high in the cells cryopreserved using the cryopreservation medium of Example 1, and very high in the cells cryopreserved using the cryopreservation medium of Comparative Example 1. was low. These results show that the cryopreservation solution of the present invention can satisfactorily cryopreserve cells while maintaining cell functions.

Since the cryopreservation solution of the present invention does not contain cytotoxic compounds such as DMSO and ethylene glycol, the cryopreservation solution of the present invention, unlike conventional cryopreservation solutions, does not expose biological samples to stress conditions. It has a remarkable effect that it can be cryopreserved while maintaining its properties. It is possible to add cytotoxic chemical substances such as DMSO and ethylene glycol at low concentrations that do not impair cell functions.

In addition, since no serum or serum-derived protein is added, there is no contamination with bacteria or viruses. It is possible to add a cytotoxic compound at a low concentration that does not impair cell functions. Also, it is possible to add proteins that do not cause infection.

<Test Example 5: Evaluation of properties (undifferentiated property) after cryopreservation using test samples of primary human mesenchymal stem cells>
The cultured primary human mesenchymal stem cells (Lonza PT2501) were suspended in the test cryopreservation medium (serum-free) of Example 1 and Comparative Example 1 at a concentration of 1×10 ⁶ cells/mL. Cell suspensions containing each test cryopreservative were then frozen in a -80°C freezer in a slow cell freezer (Nalgene® Mr. Frosty). The frozen cell suspension containing each test cryopreservation medium was cryopreserved at −80° C. for 6 months. After 6 months, the cell suspension containing each test cryopreservation medium was removed and rapidly thawed in a 37°C hot bath. In the cell suspension containing each test cryopreservation medium 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 representative 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 observed in cells cryopreserved in the cryopreservation medium of Example 1 and cryopreserved in the cryopreservation medium of Comparative Example 1. higher than cells. Therefore, it can be seen that the cells cryopreserved in the cryopreservation solution of Example 1 maintain the expression of the undifferentiated marker. That is, the cells cryopreserved with the cryopreservation solution of Example 1 can maintain an undifferentiated state. According to the cryopreservation of the present invention, it can be seen that the influence on the state of differentiation seen in the case of preservation with the cryopreservation solution of Comparative Example 1 can be reduced.

These results show that the cryopreservation method using the cryopreservation solution of the present invention can be suitably applied to the cryopreservation of stem cells, for which it is important to cryopreserve in an undifferentiated state.

<Test Example 6: Evaluation of cryopreservation solution containing various polysaccharides>
Suspend the cultured primary canine mesenchymal stem cells (cyagen C160) at a concentration of 1×10 ⁶ cells/mL in the test cryopreservation medium (serum-free) of Examples 1, 3 to 9 and Experimental Example 1. (However, the concentration of the hyaluronic acid sample in the test cryopreservation solution of Example 1 was 20 w/v%). Cell suspensions containing each test cryopreservative were then frozen in a -80°C freezer in a slow cell freezer (Nalgene® Mr. Frosty). After cryopreservation for 1 day, the cell suspension containing the cryopreservation medium for each test was taken out and rapidly thawed in a 37°C hot bath. The cell viability of the cell suspension containing each test cryopreservation medium after thawing was evaluated by trypan blue staining. The results are shown in FIG.

From FIG. 7, regardless of the type of polysaccharide, not only hyaluronic acid but also other polysaccharides such as pullulan, any polysaccharide having a predetermined molecular weight is suitable as a cryoprotectant in a cryopreservation solution. It can be seen that it exhibits a significant cytoprotective effect. In addition, dextran showed only a low cytoprotective effect as a cryopreservation agent. Moreover, the cell preserving effect was further improved by using a combination of polysaccharides and fragments, which are cleavage products of polysaccharides. When fragments are added, the prepared cryopreservation solution may be acidic, but in such cases, neutralizing the cryopreservation solution by pH adjustment will increase the cytoprotective effect. Got. It is believed that the pH adjustment provided more suitable conditions for cell survival. In addition, when the fragment was added, it was preferable to add it in an amount of 1 to 5 w/v% relative to the test sample, which is a polysaccharide, in order to further improve the cytoprotective effect. Even if the amount of fragment to be added was increased to more than 10 w/v % relative to the test sample, no further effect on the effect of improving cell viability was observed.

Moreover, from the results shown in FIG. 7, it was found that the cryopreservation solution of the present invention exhibits a good cryoprotective effect not only on human but also on canine mesenchymal stem cells.

<Test Example 7: Evaluation of the effect of adding various fragments>
The cultured primary canine mesenchymal stem cells (cyagen C160) were suspended in the test cryopreservation medium (serum-free) of Examples 10 and 11 at a concentration of 1×10 ⁶ cells/mL. Cell suspensions containing each test cryopreservative were then frozen in a -80°C freezer in a slow cell freezer (Nalgene® Mr. Frosty). After cryopreservation for 1 day, the cell suspension containing the cryopreservation medium for each test was taken out and rapidly thawed in a 37°C hot bath. The cell viability of the cell suspension containing each test cryopreservation medium after thawing was evaluated by trypan blue staining immediately after thawing. The results are shown in FIG.

As shown in FIG. 8, a production example of a viscosity average molecular weight of 1000 obtained by glucuronic acid and subcritical treatment than sucrose that is sometimes added to conventional cryopreservation solutions, which is considered preferable from the viewpoint of water retention A higher cytoprotective effect was observed when one hyaluronic acid fragment sample was added. Based on these results, it is thought that fragments that are cleavage products of glycosaminoglycan, particularly fragments that are cleavage products of hyaluronic acid and glucuronic acid, which is a constituent monosaccharide of hyaluronic acid, are preferable as additional components to be added. be done.

<Test Example 8: Evaluation of cells in frozen cryopreservation solution (evaluation of intracellular vitrification state)>
The cultured primary canine mesenchymal stem cells (cyagen C160) were suspended in the test cryopreservation medium (serum-free) of Example 1 and Comparative Example 1 at a concentration of 1×10 ⁶ cells/mL. As a control, a control suspension was similarly prepared by suspending cells in the test sample solution of Comparative Example 2 consisting of αMEM medium containing no cryopreservative. After that, 5 μL of the cell suspension containing the cryopreservation solution for each test and the control suspension were added to a hard glass sample plate (16φ×0.12 mm), and a hard glass cover glass (12φ×0.12 mm) was added. ), cooled to −80° C. at 5° C./min on a linKam microscope cooling stage (THMS600), and images were taken at −80° C. with a transmitted light microscope (Olympus BX53). The results are shown in Figures 9A-C.

FIG. 9A shows the results of the control suspension, and it can be seen that the cells darkened due to the generation of ice crystals in the cells and diffuse reflection of light in the medium alone. FIG. 9B shows the results of Comparative Example 1, in which DMSO is the test sample. Also in FIG. 9B, the cells are darkened, indicating the formation of micro ice crystals. 9C shows the results of the cryopreservation solution containing the test sample of Example 1. FIG. It can be seen that the cells are bright and vitrified in an amorphous state inside the cells.

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

<Test Example 9: Evaluation of intracellular vitrified state using brightness difference>
The cultured primary canine mesenchymal stem cells (cyagen C160) were suspended in the test cryopreservation medium (serum-free) of Example 1 and Comparative Example 1 at a concentration of 1×10 ⁶ cells/mL. As a control, a control suspension was similarly prepared by suspending cells in the test sample solution of Comparative Example 2 consisting of αMEM medium containing no cryopreservative. After that, 5 μL of the cell suspension containing the cryopreservation solution for each test and the control suspension were added to a hard glass sample plate (16φ×0.12 mm), and a hard glass cover glass (12φ×0.12 mm) was added. ), cooled to −80° C. at 5° C./min on a linKam microscope cooling stage (THMS600), and images were taken at −80° C. with a transmitted light microscope (Olympus BX53). The results are shown in Figures 10A and 10B.

The difference (absolute value) between the intracellular and extracellular light and darkness in the observation image shown in FIG. 10A was analyzed using image analysis software ImageJ (https://imagej.nih.gov/ij/). In each image, the brightness of the solvent region, the brightness inside the cell, and the Munsell brightness were read under the same conditions, and the reading data for the brightness of the solvent region and the brightness inside the cell were compared with the reading data for 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 conversion). In addition, if the read value of the brightness of the intracellular region or the solvent region read by the image analysis software is darker than the read value of the Munsell brightness value 0, which is the darkest value of 0, the Munsell If the value is set to 0, and the read value of the brightness of the intracellular region or the solvent region read by the image analysis software is brighter than the reading value of the brightest Munsell brightness value of 10, for convenience, the intracellular region or the solvent region Assume that the Munsell brightness value of the region is 10.

As a result of the measurement, in the control suspension (Comparative Example 2), the Munsell brightness in the solvent region was 5, the Munsell brightness in the intracellular region was 0, and the difference in Munsell brightness between the solvent region and the intracellular region was 5. . In Comparative Example 1, the brightness of the solvent region was 4, the Munsell brightness of the intracellular region was 0, and the Munsell brightness difference was 4. In contrast, in Example 1, the Munsell brightness in the solvent region was 4, the Munsell value in the intracellular region was 2, and the Munsell brightness difference was 2 (FIG. 10B).

That is, in Example 1, the brightness of the intracellular region and the brightness of the solvent region are close. This result indicates that in the cryopreservation solution containing the test sample of Example 1, the frozen cells turned bright, that is, the inside of the cells was vitrified. The brightness difference between the intracellular region and the solvent region is desirably 3 or less. Moreover, it is desirable that the brightness value of the solvent region is greater than or equal to the brightness value of the intracellular region.

<Test Example 10: Evaluation of cell area in frozen cryopreservation solution>
The cultured primary canine mesenchymal stem cells (cyagen C160) were suspended in the test cryopreservation medium (serum-free) of Example 1 and Comparative Example 1 at a concentration of 1×10 ⁶ cells/mL. As a control, a control suspension was similarly prepared by suspending cells in a medium containing the test sample solution of Comparative Example 2 (i.e., medium only) consisting of αMEM medium containing no cryopreservative. After that, the cell suspension containing each test cryopreservation medium and the control suspension were cooled to -80 ° C. at 5 ° C./min on a linKam microscope cooling stage (THMS600), and then -50 ° C./ It was melted quickly at min. Before cooling and after melting, the area of 10 cells present in the field of view was calculated using image analysis software ImageJ. The results are shown in FIG.

As shown in FIG. 11, in Comparative Example 2 containing only the medium and Comparative Example 1 containing DMSO as the test sample, the area of cells increased after freezing, whereas the cells were frozen using the cryopreservation solution of the present invention. After that, the cells had shrunk to about 1/3. It was confirmed that the inside of the cells was well dehydrated during freezing. Desirably, the cells have shrunk to less than 1/1, 1/3.5 or more of their orthogonal projected area in the medium.

<Test Example 11: 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 added at a concentration of 1×10 ⁶ cells/mL. 1 and the test cryopreservation medium (serum-free) of Comparative Example 1. Cell suspensions containing each test cryopreservative were then frozen in a -80°C freezer in a slow cell freezer (Nalgene® Mr. Frosty). After cryopreservation for 7 days, the cell suspension containing the cryopreservation medium for each test was taken out and rapidly thawed in a 37°C hot bath. The cell viability of the cell suspension containing each test cryopreservation medium after thawing was evaluated by trypan blue staining. The results are shown in FIG.

As shown in FIG. 12, when cryopreserved using the cryopreservation medium of the present invention, all cells exhibited a high cell viability that was substantially equal to or higher than that of Comparative Example 1 in which DMSO was the test sample. Got. From these results, it was confirmed that the cryopreservation solution of the present invention can cryopreserve various types of cells with a high cell survival rate, regardless of whether the cells are primary cells or established cells, and regardless of the origin of the cells. was done.

<Test Example 12: Evaluation of cryoprotective effect in cryopreservation using saccharides with low molecular weight>
The cultured primary canine mesenchymal stem cells (cyagen C160) were dissolved in αMEM medium at a concentration of 1×10 ⁶ cells/mL, and each test sample of Production Examples 1 to 3 was dissolved at a concentration of 10 w/v%. were suspended in each test cryopreservation medium. Cell suspensions containing each test cryopreservative were then frozen in a -80°C freezer in a slow cell freezer (Nalgene® Mr. Frosty). After cryopreservation for 7 days, the cell suspension containing the cryopreservation medium for each test was taken out and rapidly thawed in a 37°C hot bath. The cell viability of the cell suspension containing each test cryopreservation medium after thawing was evaluated by trypan blue staining. The results are shown in FIG.

As shown in FIG. 13, the cell survival effect cannot be obtained only with saccharides having a molecular weight smaller than that of the polymer. A cell viability of about 10% was observed in Preparation 3, presumably due to hyaluronic acid with a molecular weight greater than 3000 that may be contained in the preparation 3 test sample.

<Test Example 13: 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 then the components of each test sample were analyzed by HPLC. As mobile phases, A solution: 16 mM NaH ₂ PO ₄ aqueous solution, B solution: 800 mM NaH ₂ PO ₄ aqueous solution, ZORBAX NH ₂ (manufactured by Agilent Technologies, column size φ4.6 × 250 mm, particle size 5 μm). 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 mobile phase B concentration 0% (0 min)→mobile phase B concentration 100% (60 min). The results are shown in Figures 14A-C. In addition, as a standard, at a concentration of 0.2 wt%, disaccharide HA02, tetrasaccharide HA04, hexasaccharide HA06, octasaccharide HA08, and decasaccharide HA10 (all manufactured by Izuron Co., Ltd.) Aqueous solutions each containing hyaluronic acid oligosaccharides were prepared and subjected to HPLC analysis in the same manner. The results are shown in FIG. 14D.

Under these HPLC analysis conditions, the monosaccharide has a retention time of 3.5 minutes, and the disaccharide has a retention time of around 9 minutes, as shown in FIG. 14D. FIG. 14A shows the analysis results of a test sample containing the hyaluronic acid cleavage product of Production Example 1 and having a viscosity-average molecular weight of 1,000. , a peak corresponding to the disaccharide with 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 is considered to contribute to the effect of improving the cell viability.

<Test Example 14: HPLC analysis of test sample of Example 1>
For the test sample of Example 1 (hyaluronic acid sample having a viscosity average molecular weight of about 10,000 obtained by subcritical treatment), using the same conditions as the HPLC analysis of the test samples of Production Examples 1 to 3 described above, HPLC analysis was performed. The results are shown in FIG.

Similarly, when compared with FIG. 14D, it can be seen that peaks corresponding to monosaccharides and disaccharides are also observed in the test sample of Example 1 at retention times of around 3.5 minutes and around 9 minutes, respectively. That is, it is considered that the test sample of Example 1 also contains such monosaccharide and disaccharide components, and this is the reason why the high cell survival rate effect is obtained.

<Test Example 15: Evaluation of cell viability using a test sample having no polysaccharide structure>
Cell viability after cryopreservation was evaluated using the test cryopreservation medium containing gelatin of Comparative Example 3 instead of the polysaccharide of the present invention. The cultured primary canine mesenchymal stem cells (cyagen C160) were suspended in the test cryopreservation medium (serum-free) of Comparative Examples 1 and 3 at a concentration of 1×10 ⁶ cells/mL. Cell suspensions containing each test cryopreservative were then frozen in a -80°C freezer in a slow cell freezer (Nalgene® Mr. Frosty). After cryopreservation for 1 day, the cell suspension containing the cryopreservation medium for each test was taken out and rapidly thawed in a 37°C hot bath. The cell viability of the cell suspension containing each test cryopreservation medium after thawing was evaluated by trypan blue staining. The results are shown in FIG.

As shown in FIG. 16, when gelatin was used as the cryopreservation agent, only a significantly lower cell survival rate was obtained than in Comparative Example 1 in which DMSO was the cryopreservation agent. Therefore, it can be seen that the cryopreservation solution of the present invention exhibits a high cell viability effect by containing the polysaccharide of the present invention.

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

When compared at the same concentration (10 w/v%) as the cryopreservation agent in the cryopreservation solution, it can be seen that hyaluronic acid having a six-membered ring structure is superior in cell preservation effect to fructan. FIG. 17 shows a graph of Table 5 of JP-A-2012-235728 (concentration on the horizontal axis is w/v%). The viability of cells is high, and in the case of five-membered ring structure fructans, it cannot be said that the cryopreservation solution exhibits sufficient functions unless the concentration is high.

From the above results, the cryopreservation solution for a biological sample containing the polysaccharide of the present invention stably vitrifies the inside of the cell, so that a cell-permeable and cytotoxic compound such as DMSO and ethylene glycol, and / Alternatively, it can be seen that there is a remarkable effect that a biological sample can be cryopreserved with a high cell viability without basically requiring the addition of serum or serum-derived proteins. Cells are well protected and their properties are maintained. In addition, when the cryopreservation solution for biological samples of the present invention further contains a polysaccharide fragment component, the vitrified state of cells is further stabilized, and a higher cell viability can be obtained.

Claims (35)

A cryopreservation solution for a biological sample containing, in a solvent, a polysaccharide or a salt thereof having a viscosity average molecular weight of more than 3000 and not more than 500000 and having a six-membered ring structure in the main chain. The cryopreservation solution according to claim 1, wherein the polysaccharide is a polysaccharide other than dextran. 3. The cryopreservation medium according to claim 1 or 2, wherein the polysaccharide comprises pentose, hexose or uronic acid or a combination thereof as repeating units. The cryopreservation solution according to any one of claims 1 to 3, wherein the polysaccharide contains amino sugars as repeating units. The cryopreservation solution according to any one of claims 1 to 4, wherein the polysaccharide has unmodified functional groups. The cryopreservation solution according to any one of claims 1 to 5, wherein the polysaccharide has a viscosity average molecular weight of 10,000 or more. The cryopreservation solution according to claim 1, wherein said polysaccharide is hyaluronic acid or pullulan. The cryopreservation solution according to any one of claims 1 to 7, wherein the concentration of the polysaccharide in the cryopreservation solution is 5 w/v% or more and 20 w/v% or less. The cryopreservation solution according to any one of claims 1 to 8, which is an intracellular impermeable cryopreservation solution. The cryopreservation solution according to any one of claims 1 to 9, wherein the biological sample is a cell, tissue, or tissue-like material that is a membrane or an aggregate. The cryopreservation solution according to claim 10, wherein the biological sample is mesenchymal stem cells, blood cells, endothelial cells, or tissue for transplantation. The cryopreservation solution according to claim 10, wherein the biological sample is sperm, egg, or fertilized egg. A method of freezing a biological sample, comprising cooling and freezing a biological sample using the cryopreservation solution according to any one of claims 1 to 12 by slow freezing at a cooling rate of 10°C/min or less. A step of including a biological sample in a cryopreservation solution containing a polysaccharide or a salt thereof having a viscosity average molecular weight of more than 3000 and not more than 500000 and having a six-membered ring structure in the main chain in a solvent;
subjecting the cryopreservation solution containing the biological sample to freezing;
and a step of preserving the cryopreservation solution containing the biological sample at a temperature of −27° C. or less. 15. The method of cryopreserving a biological sample according to claim 14, wherein the polysaccharide is a polysaccharide other than dextran. 16. The biological sample cryopreservation method according to claim 14 or 15, wherein the step of including the biological sample in the cryopreservation solution is performed before cooling the biological sample. The biological sample cryopreservation method according to any one of claims 14 to 16, wherein the biological sample is a cell, a tissue, or a tissue-like substance that is a membrane or an aggregate. 18. The biological sample cryopreservation method according to claim 17, wherein the biological sample is a sperm, an egg, or a fertilized egg. A method of preserving a biological sample, comprising:
The biological sample has a viscosity average molecular weight of more than 3000 and less than or equal to 500,000, in the presence of a polysaccharide or a salt thereof containing a six-membered ring structure in the main chain, and stored at -27 ° C. or less,
The storage reduces the viability of the biological sample by less than 5% relative to the viability of the biological sample immediately prior to storage when the biological sample is stored in the presence of the polysaccharide or salt thereof for a period of at least 5 months. A method of preserving a biological sample, characterized in that the preservation exhibits The storage reduces the viability of the biological sample by less than 10% relative to the viability of the biological sample immediately prior to storage when the biological sample is stored in the presence of the polysaccharide or salt thereof for a period of at least 6 months. 20. The method of preserving a biological sample according to claim 19, wherein the preservation exhibits When the biological sample is stored at 4° C. for 1 day after being frozen at -27° C. or lower and then thawed, the survival rate is reduced by less than 5% based on the survival rate of the biological sample immediately after thawing. 20. The method of preserving a biological sample according to claim 19, characterized in that The method for preserving a biological sample according to any one of claims 19 to 21, wherein said biological sample is a cell. 23. The method of preserving a biological sample according to claim 22, wherein said biological sample is mammalian cells. 24. The method of preserving a biological sample according to claim 23, wherein the biological sample is mammalian mesenchymal stem cells, mammalian blood cells, or mammalian endothelial cells. Use of a polysaccharide having a viscosity average molecular weight of more than 3000 and not more than 500000 and containing a six-membered ring structure in its main chain or a salt thereof for preserving a biological sample at a temperature of -27°C or less. 26. Use according to claim 25, wherein said polysaccharide is a polysaccharide other than dextran. Use of a polysaccharide having a viscosity-average molecular weight of more than 3000 and not more than 500000 and containing a six-membered ring structure in its main chain or a salt thereof for cryopreserving a biological sample. 28. Use according to claim 27, wherein said polysaccharide is a polysaccharide other than dextran. A cryopreservation agent for biological samples, comprising a polysaccharide or a salt thereof having a viscosity average molecular weight of more than 3000 and not more than 500000 and containing a six-membered ring structure in the main chain. The cryopreservation agent for biological samples according to claim 29, wherein the polysaccharide is a polysaccharide other than dextran. 31. The cryopreservation agent for a biological sample according to claim 29 or 30, wherein the polysaccharide contains pentose, hexose, uronic acid, or a combination thereof as repeating units. The cryopreservation agent for a biological sample according to any one of claims 29 to 31, wherein the polysaccharide contains an amino sugar as a repeating unit. The cryopreservation agent for a biological sample according to any one of claims 29 to 32, wherein the polysaccharide has unmodified functional groups. The cryopreservation agent for biological samples according to any one of claims 29 to 33, wherein the polysaccharide has a viscosity average molecular weight of 10,000 or more. The cryopreservation agent for biological samples according to any one of claims 29 to 34, wherein the polysaccharide is hyaluronic acid or pullulan.

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