Cryopreservation liquid for biological samples and cryopreservation method for biological samples

Abstract

This cryopreservation liquid contains an aqueous solvent, a water-soluble polymer, and at least one cryoprotective support substance selected from the group consisting of glycine, alanine, valine, asparagine, isoleucine, glutamine, histidine, proline, hydroxyproline, and taurine. The viscosity of the cryopreservation liquid at a shear rate of 100 s-1 at 23°C is 30 mPa·s or less.

Description

Cryopreservation solution for biological samples and method for freezing and preserving biological samples

[0001] The present invention relates to a cryopreservation solution for biological samples and a method for cryopreserving biological samples using the same.

[0002] Cryopreservation of biological samples such as cells and tissues is an important technology not only in the field of regenerative medicine but also in various fields such as livestock farming and food production. For example, in the field of regenerative medicine, stem cells collected from living organisms, such as bone marrow-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells, are grown in large quantities before use. In this case, it is common to freeze and store surplus cells, and then thaw and use them when needed.

[0003] During cell cryopreservation, the growth of ice crystals within cells can damage cell membranes and intracellular structures, and can denature cellular proteins, resulting in fatal damage to the cells. To prevent such cell damage, methods for cryopreserving cells, such as slow freezing and vitrification, are used. Slow freezing involves slowly cooling cells in a solution containing a low concentration of cryoprotectant, which dehydrates and freezes the cells, preventing the formation of intracellular ice crystals. Vitrification involves rapidly cooling cells in a solution containing a high concentration of cryoprotectant, preventing the formation of intracellular and extracellular ice crystals.

[0004] Dimethyl sulfoxide (DMSO) is widely used as a cryoprotectant. However, DMSO is known to have properties that adversely affect cells (especially stem cells), such as cytotoxicity and differentiation induction. For this reason, cryopreservation solutions with low or no DMSO concentration have been developed, and various solutions have been proposed (see, for example, Patent Document 1).

[0005] Patent Document 1 describes a cryopreservation solution for blood cells that contains a solvent, a polymer (e.g., hyaluronic acid) with a viscosity-average molecular weight of more than 3,000 and not more than 500,000, a sugar with a viscosity-average molecular weight of not more than 3,000, and a polyhydric alcohol (e.g., glycerol). It is explained that the cryopreservation solution described in Patent Document 1 can cryopreserve blood cells with a high survival rate even without containing DMSO.

[0006] Japanese Patent Application Laid-Open No. 2022-104329

[0007] However, the cryopreservation solution described in Patent Document 1 uses a large amount of viscous materials such as hyaluronic acid and glycerol, which makes it prone to foaming during pipetting and leaves room for improvement in terms of operability. Meanwhile, according to preliminary experiments by the present inventors, reducing the concentrations of hyaluronic acid, glycerol, etc. in the cryopreservation solution described in Patent Document 1 results in a decrease in cell viability.

[0008] Therefore, an object of the present invention is to provide a cryopreservation solution for biological samples that is easy to use and that allows cryopreservation of biological samples with a high survival rate even without containing DMSO. Another object of the present invention is to provide a method for cryopreserving biological samples using the cryopreservation solution.

[0009] The present invention relates to the following cryopreservation solution for biological samples and cryopreservation method for biological samples.

[0010] [1] A method for preparing a solution containing an aqueous solvent, a water-soluble polymer, and at least one amino acid selected from the group consisting of glycine, alanine, valine, asparagine, isoleucine, glutamine, histidine, proline, and hydroxyproline, wherein the concentration of the amino acid is more than 0.5 w / v%, and the solution is simmered at 23°C for 100 seconds. －１ [2] A cryopreservation solution for biological samples, the viscosity of which at a shear rate of 23°C is 30 mPa s or less. －１[3] A cryopreservation solution for biological samples according to [1] or [2], wherein the viscosity is 5 mPa·s or more. [4] A cryopreservation solution for biological samples according to any one of [1] to [3], wherein in differential scanning calorimetry, an exothermic peak is observed in the range of −30 to −10°C in a DSC curve obtained during cooling from 20°C to −80°C, and the heat of fusion calculated from the exothermic peak is 250 J / g or less. [5] A cryopreservation solution according to any one of [1] to [4], wherein the water-soluble polymer comprises at least one selected from the group consisting of hyaluronic acid and a salt thereof, and alginic acid and a salt thereof. [6] A cryopreservation solution according to [1], wherein the amino acid comprises proline. [7] A cryopreservation solution according to [6], wherein the concentration of proline is within the range of 1 to 10 w / v%. [8] The cryopreservation solution according to [2], wherein the concentration of taurine is within the range of 0.01 to 5 w / v%. [9] The cryopreservation solution according to [2] or [8], further comprising proline.

[10] The cryopreservation solution according to any one of [1] to [9], wherein the concentration of dimethyl sulfoxide is 10 w / v% or less.

[11] The cryopreservation solution according to any one of [1] to

[10] , wherein the total concentration of a polyhydric alcohol selected from the group consisting of ethylene glycol, propylene glycol, and glycerol is 30 v / v% or less.

[12] The cryopreservation solution according to any one of [1] to

[11] , wherein the biological sample is a cell.

[13] A method for cryopreserving a biological sample, comprising the steps of: incorporating a biological sample in the cryopreservation solution according to any one of [1] to

[12] ; and freezing the cryopreservation solution containing the biological sample.

[0011] According to the present invention, a cryopreservation solution for biological samples can be provided that is DMSO-free and allows for cryopreservation of biological samples with a high survival rate, and is easy to handle.Furthermore, according to the present invention, a cryopreservation method for biological samples using the cryopreservation solution can be provided.

[0012] Figure 1 is a graph showing the relationship between the concentration of hyaluronic acid solution and reduced viscosity. Figure 2 is a graph showing the relationship between the shear rate and viscosity of cryopreservation solutions. Figures 3A and 3B are graphs showing the survival rate of cells after thawing for each cryopreservation solution.

[0013] Hereinafter, embodiments of the present invention will be specifically described, but the present invention is not limited to these.

[0014] In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits. In numerical ranges described in stages in this specification, the upper or lower limit described in a certain numerical range may be replaced with the upper or lower limit of another numerical range described in stages.

[0015] [Cell Cryopreservation Solution] The cryopreservation solution for biological samples according to the present invention (hereinafter also referred to as "cryopreservation solution") comprises an aqueous solvent, a water-soluble polymer, and a cryoprotective support substance. The cryoprotective support substance comprises at least one amino acid selected from the group consisting of glycine, alanine, valine, asparagine, isoleucine, glutamine, histidine, proline, hydroxyproline, and taurine.

[0016] The type of biological tissue to be cryopreserved is not particularly limited as long as it contains cells. Examples of biological tissue include cells (e.g., stem cells, germ cells, somatic cells, fertilized eggs, etc.), early embryos, tissues, organized cell structures, and tissue-like substances (e.g., membrane-like substances and aggregates containing a large number of cells, etc.). The species from which the biological tissue is derived is also not particularly limited.

[0017] The viscosity of the cryopreservation solution according to the present invention is 30 mPa·s or less. By setting the viscosity at 30 mPa·s or less, foaming is less likely to occur during pipetting, improving operability. From the same perspective, the viscosity of the cryopreservation solution is preferably 15 mPa·s or less. On the other hand, the lower limit of the viscosity of the cryopreservation solution is not particularly limited, but from the viewpoint of appropriately lowering the fluidity of water to reduce ice crystal formation during freezing and thereby further increasing cell viability, 5 mPa·s or more is preferred, 8 mPa·s or more is more preferred, and 10 mPa·s is even more preferred. That is, the viscosity of the cryopreservation solution is 0 mPa·s or more and 30 mPa·s or less, preferably 5 mPa·s or more and 15 mPa·s or less.

[0018] In this specification, the "viscosity of the cryopreservation solution" refers to the viscosity measured at 100 s using a rheometer. －１ The viscosity of the cryopreservation solution at 23°C is measured at a shear rate of 100 s. The viscosity of the cryopreservation solution can be adjusted to within the above range by adjusting the type and concentration of each component (particularly the viscosity average molecular weight and concentration of the water-soluble polymer). The viscosity of the cryopreservation solution according to the present invention is measured at a shear rate of 100 s. －１ ~1000s －１ (See FIG. 2.) The viscosity of the cryopreservation solution can be adjusted, for example, by the concentration and molecular weight of the water-soluble polymer in the cryopreservation solution, the total concentration of the polyhydric alcohol, etc. For example, the smaller the molecular weight of the water-soluble polymer and the lower the concentration of the water-soluble polymer and the total concentration of the polyhydric alcohol in the cryopreservation solution, the lower the viscosity of the cryopreservation solution.

[0019] (Aqueous Solvent) The type of aqueous solvent is not particularly limited as long as it allows for appropriate cryopreservation of biological samples. The aqueous solvent may be, for example, an isotonic solution in which the salt concentration and sugar concentration are adjusted using sodium ions, potassium ions, calcium ions, etc. to approximately match the osmotic pressure of body fluids or cellular fluids. Examples of aqueous solvents include water; physiological saline; buffered physiological saline solutions such as phosphate buffered saline (PBS), Dulbecco's phosphate buffered saline, Tris buffered saline (TBS), and HEPES-buffered saline; balanced salt solutions such as Hank's balanced salt solution (HBSS); Ringer's solutions such as Ringer's solution, lactate Ringer's solution, acetate Ringer's solution, and bicarbonate Ringer's solution; and commercially available oral rehydration solutions. Furthermore, as long as the effects of the present invention are not impaired, the aqueous solvent may contain other optional components, such as isotonic agents, chelating agents, solubilizers, pH adjusters, and additives commonly used in cell culture media.

[0020] The aqueous solvent may be a cell culture medium such as a commercially available medium or a basal medium such as D-MEM, E-MEM, αMEM, RPMI-1640 medium, Ham's F-12, Ham's F-10, M-199, etc. The cryopreservation solution according to the present invention also includes a cell culture or cell suspension to which a water-soluble polymer and a cryoprotective support substance have been added at predetermined concentrations.

[0021] (Water-Soluble Polymer) The water-soluble polymer is a polymer having a predetermined molecular weight and numerous hydrophilic groups. The water-soluble polymer may be in a salt state. When a biological sample is cryopreserved using the cryopreservation solution of the present invention, water molecules of the solvent are trapped within the matrix formed by the water-soluble polymer during the cooling process of the cryopreserved biological sample. Because the polymer chains contain hydrophilic groups, the molecular motion of water is restricted during cooling, allowing the water to solidify and / or freeze in a vitrified state without crystallizing. Because the action of these polymer chains also dehydrates and vitrifies the intracellular contents, cryopreservation using the cryopreservation solution of the present invention does not require the increased concentration of solutes (cryoprotectants) or the increased cooling rate required in conventional vitrification methods. In the present invention, the action of the polymer chains suppresses intracellular ice crystal formation, thereby reducing the osmotic shock experienced by cells during freezing, a problem associated with conventional vitrification methods that utilize the osmotic pressure difference between the inside and outside of the cells to dehydrate and vitrify the interior of the cells. Furthermore, since recrystallization does not occur during thawing of frozen cells, thawing damage to the cells is thought to be minimal.

[0022] A water-soluble polymer is a polymer containing a repeating unit of a monomer having a hydrophilic group. Examples of the hydrophilic group include a hydroxy group, a carboxy group, and salts thereof. The water-soluble polymer may also contain a repeating unit of a nitrogen-containing monomer having an optionally substituted amino group or an optionally substituted amide group. Furthermore, the water-soluble polymer preferably has a hydroxy group at an equatorial position within its structure. This is believed to enable the solvent water to be more effectively trapped within the matrix formed by the polymer chains during freezing.

[0023] The monomer having a hydrophilic group is, for example, a sugar residue. In this case, the water-soluble polymer may be a polymer containing repeating units of sugar residues linked by glycosidic bonds or derivatives thereof. The sugar residue may be, but is not limited to, a monosaccharide or a monosaccharide in which the hydroxyl and / or hydroxymethyl groups of the monosaccharide are substituted with a substituent. Examples of monosaccharides include triose, tetrose, pentose, hexose, and heptose. Examples of pentoses include ribose, arabinose, xylose, lyxose, xylulose, ribulose, and deoxyribose. Examples of hexoses include glucose, mannose, galactose, fructose, sorbose, tagatose, fucose, fuculose, and rhamnose. Examples of substituents include a carboxy group, an amino group, an N-acetylamino group, a sulfoxy group, a methoxycarbonyl group, and a carboxymethyl group.

[0024] An example of a monosaccharide substituted with a carboxy group is uronic acid. Examples of uronic acids include glucuronic acid, iduronic acid, mannuronic acid, and galacturonic acid. An example of a monosaccharide substituted with an amino group is an amino sugar. Examples of amino sugars include glucosamine, galactosamine, mannosamine, and muramic acid. Examples of monosaccharides substituted with an N-acetylamino group are N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, and N-acetylmuramic acid. An example of a monosaccharide substituted with a sulfooxy group is galactose-3-sulfate. Examples of monosaccharides with multiple substituents are N-acetylglucosamine-4-sulfate, iduronic acid-2-sulfate, glucuronic acid-2-sulfate, N-acetylgalactosamine-4-sulfate, neuraminic acid, and N-acetylneuraminic acid.

[0025] For example, the water-soluble polymer is a polymer containing the above-mentioned monosaccharides as repeating units. Specifically, the water-soluble polymer may be a polymer containing optionally substituted pentose, hexose, or uronic acid, or a combination thereof, as repeating units. The water-soluble polymer may also be an alternating copolymer of a monomer having a hydrophilic group and a nitrogen-containing monomer. The nitrogen-containing monomer is, for example, an amino sugar. In this case, the water-soluble polymer may be a glycosaminoglycan. The water-soluble polymer may also be a sulfated polysaccharide in which one or more hydroxyl groups are substituted with sulfoxy groups. Examples of water-soluble polymers include hyaluronic acid, alginic acid, dextran, pullulan, chondroitin sulfate, and salts thereof.

[0026] As described above, the water-soluble polymer may be in the form of a salt. The salt of the water-soluble polymer may be, for example, a metal salt, a halogen salt, or a sulfate salt. As the metal salt, a salt of an alkali metal or an alkaline earth metal is preferred. Examples of alkali metals and alkaline earth metals include sodium, potassium, and calcium. Examples of halogens include chlorine and bromine.

[0027] The hydrophilic groups are preferably unmodified, or if modified, the proportion of the hydrophilic groups is 50% or less of the total number of hydrophilic groups, i.e., no substituents are introduced into the polymer chain, or if introduced, the proportion of the hydrophilic groups is 50% or less of the total number of hydrophilic groups.Hydrophilic groups, particularly hydroxyl groups, amino groups, and carboxyl groups, are presumed to contribute to the protection of frozen cells and the vitrification of the solvent, so it is thought that not modifying these functional groups is advantageous for improving the survival rate of cells after thawing.

[0028] The water-soluble polymer may be a naturally occurring polymer compound, a chemically synthesized polymer compound, or a commercially available polymer compound.The water-soluble polymer to be incorporated into the cryopreservation solution may be a polymer compound with a larger molecular weight, whose molecular weight has been adjusted by hydrolysis, enzyme treatment, subcritical treatment, or other treatment.From the viewpoint of reducing the cytotoxicity of the cryopreservation solution, the water-soluble polymer is preferably a biological component, and is preferably at least one selected from the group consisting of hyaluronic acid and its salt, and alginic acid and its salt (for example, sodium alginate).

[0029] The monomer may be a naturally occurring compound, a compound obtained by modifying or substituting a naturally occurring compound, or a chemically synthesized compound. From the viewpoint of reducing the cytotoxicity of the cryopreservation solution, the monomer is preferably a biological component.

[0030] The viscosity-average molecular weight of the water-soluble polymer is not particularly limited as long as it can reduce the viscosity of the cryopreservation solution to 30 mPa·s or less. Generally, the term "polymer" refers to a molecule with a molecular weight exceeding 10,000, but the viscosity-average molecular weight of the water-soluble polymer in the cryopreservation solution of the present invention may be 10,000 or less. For example, the viscosity-average molecular weight of the water-soluble polymer is preferably within the range of 5,000 to 200,000, more preferably within the range of 5,000 to 100,000, and particularly preferably within the range of 5,000 to 50,000. If the viscosity-average molecular weight of the water-soluble polymer is less than 5,000, vitrification may not occur satisfactorily. Furthermore, if the viscosity-average molecular weight of the water-soluble polymer is greater than 200,000, the viscosity of the cryopreservation solution may exceed 30 mPa·s, potentially reducing the operability of the cryopreservation solution.

[0031] The viscosity average molecular weight of the water-soluble polymer is calculated by the following method and formula.

[0032] (1) A water-soluble polymer sample is dissolved in a 0.2 M aqueous sodium chloride solution (standard solution) to prepare a stock solution. If the water-soluble polymer sample is in solution, the solids obtained by removing the solvent from the solution are used as the water-soluble polymer sample. If the water-soluble polymer-containing sample is a mixed sample containing multiple types of compounds, the water-soluble polymer is separated and used as the water-soluble polymer sample. However, even if the water-soluble polymer contains impurities, the mixture may also be used as the water-soluble polymer sample as long as the impurities (e.g., metal salts) do not affect the viscosity. Furthermore, if the type of water-soluble polymer is unknown, the type of water-soluble polymer is identified using HPLC, LC-MS, LC-IR, or the like. The viscosities of the standard solution and the stock solution are measured, and it is preferable to adjust the relative viscosity of the stock solution to approximately 2.0 to 2.4.

[0033] (2) The stock solution is diluted stepwise with the standard solution to prepare diluted solutions with different concentrations.

[0034] (3) Using an Ubbelohde viscometer at a temperature of 30°C, measure the flow time of each of the standard solution and the diluted solution.

[0035] (4) For each diluted solution, calculate the reduced viscosity I (dL / g) using the following formula 1. In formula 1, t is the outflow time (seconds) of the diluted solution, and t ０ is the elution time of the standard solution (seconds), and c is the concentration of the diluent (g / dL).

[0036] (5) The relationship between the concentration of the water-soluble polymer and the reduced viscosity of the water-soluble polymer is plotted, and an approximate straight line is drawn. The intercept of the approximate straight line (water-soluble polymer concentration = 0 g / dL) is taken as the intrinsic viscosity η (dL / g).

[0037] (6) Calculate the viscosity average molecular weight M (dL / g) of the water-soluble polymer using the following formula 2 (Mark Hoeing-Sakurada formula): In formula 2, η is the intrinsic viscosity (dL / g), and K and α are constants.

[0038] K and α are values ​​that vary depending on the type of water-soluble polymer, and 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, Incorporated Association). For example, in the case of hyaluronic acid, K = 3.6 × 10 in the above formula 2. －４ and α = 0.78 to calculate the viscosity average molecular weight M. In the case of sodium alginate, K = 3.62 × 10 in the above formula 2. －４ and α=1.2 to determine the viscosity average molecular weight M.

[0039] The concentration of the water-soluble polymer in the cryopreservation solution is not particularly limited as long as it allows cells to be appropriately cryopreserved and the viscosity of the cryopreservation solution to be 30 mPa·s or less. For example, the concentration of the water-soluble polymer is 0.1 w / v% or more and less than 10 w / v%. If the concentration is less than 0.1 w / v%, the solvent portion may not be vitrified satisfactorily. If the concentration is more than 10 w / v%, the viscosity of the cryopreservation solution may increase, which may deteriorate its operability. The concentration of the water-soluble polymer may be within the range of 0.5 to 8 w / v%, or within the range of 3 to 6 w / v%.

[0040] (Cryoprotective Support Substance) A cryoprotective support substance is a substance that can form ice nuclei in an extracellular solution at a temperature higher than the temperature at which intracellular water freezes. The cryopreservation solution according to the present invention cannot contain a large amount of water-soluble polymers because it has a viscosity of 30 mPa·s or less. Preliminary experiments by the present inventors showed that reducing the concentration of water-soluble polymers in the cryopreservation solution reduced cell viability. However, the present inventors found that adding a specific cryoprotective support substance can suppress the reduction in cell viability even when the concentration of water-soluble polymers in the cryopreservation solution is reduced.

[0041] As described above, the cryoprotective support substance includes at least one selected from the group consisting of glycine, alanine, valine, asparagine, isoleucine, glutamine, histidine, proline, hydroxyproline, and taurine. For example, the cryoprotective support substance may be at least one amino acid selected from the group consisting of glycine, alanine, valine, asparagine, isoleucine, glutamine, histidine, proline, and hydroxyproline. Among these amino acids, proline is preferred. The cryoprotective support substance may also be taurine. The cryopreservation solution may contain only one of the above compounds, or two or more of them. For example, the cryopreservation solution may contain both the above amino acid (e.g., proline) and taurine.

[0042] The concentration of the cryoprotective support substance is not particularly limited as long as it can adequately exert its effects. For example, when the cryoprotective support substance is an amino acid (e.g., proline), the concentration of the cryoprotective support substance (amino acid) is preferably greater than 0.5 w / v%, more preferably in the range of 1 to 10 w / v%, even more preferably in the range of 3 to 10 w / v%, and particularly preferably in the range of 5 to 10 w / v%. In other embodiments, the concentration of the cryoprotective support substance (amino acid) may be in the range of 1 to 7.5 w / v%. When multiple types of amino acids are included as the cryoprotective support substance, the above concentration refers to the total concentration of the amino acids. On the other hand, when the cryoprotective support substance is taurine, the concentration of the cryoprotective support substance (taurine) is not particularly limited, but is preferably in the range of 0.01 to 5 w / v%, more preferably in the range of 0.5 to 2 w / v%, and particularly preferably about 1 w / v%.

[0043] (Other Components) The cryopreservation solution according to the present invention may further contain other optional components as long as they do not impair the effects of the present invention.

[0044] For example, the cryopreservation solution may contain a cell membrane-impermeable cryoprotectant such as a sugar or dextran. Examples of sugars include dextrose, mannose, galactose, fructose, raffinose, lactose, sucrose, maltose, glucose, sorbitol, mannitol, and trehalose. The cryopreservation solution may contain such a cell membrane-impermeable cryoprotectant at a concentration of, for example, about 0.1 to 10 w / v %.

[0045] On the other hand, it is preferable that the cryopreservation solution is substantially free of DMSO. The concentration of dimethyl sulfoxide (DMSO) in the cryopreservation solution is preferably within the range of 0 to 10 w / v%, more preferably within the range of 0 to 5 w / v%, even more preferably within the range of 0 to 1 w / v%, even more preferably within the range of 0 to 0.5 w / v%, and particularly preferably within the range of 0 to 0.1 w / v%. By reducing the DMSO concentration, problems such as DMSO's cytotoxicity and differentiation induction can be resolved. The concentration of dimethyl sulfoxide (DMSO) in the cryopreservation solution may be 0.1 to 10 w / v%, 0.1 to 5 w / v%, 0.1 to 1 w / v%, or 0.1 to 0.5 w / v%.

[0046] Furthermore, the cryopreservation solution may be substantially free of polyhydric alcohols such as ethylene glycol, propylene glycol, and glycerol. Polyhydric alcohols are used as cryoprotectants to suppress intracellular ice crystal formation, but the cryopreservation solution of the present invention is capable of cryopreserving cells with a high survival rate even without polyhydric alcohols. For example, by omitting the addition of toxic polyhydric alcohols such as ethylene glycol, the safety of the cryopreservation solution for users can be improved. Furthermore, by omitting the addition of viscous polyhydric alcohols such as glycerol, the operability of the cryopreservation solution can be improved. The total concentration of polyhydric alcohols selected from the group consisting of ethylene glycol, propylene glycol, and glycerol in the cryopreservation solution is preferably within the range of 0 to 30 v / v%, more preferably within the range of 0 to 10 v / v%, even more preferably within the range of 0 to 5 v / v%, even more preferably within the range of 0 to 1 v / v%, even more preferably within the range of 0 to 0.5 v / v%, and particularly preferably within the range of 0 to 0.1 v / v%. The total concentration of the polyhydric alcohol selected from the group consisting of ethylene glycol, propylene glycol, and glycerol in the cryopreservation solution may be 0.1 to 30 v / v%, 0.1 to 10 v / v%, 0.1 to 5 v / v%, 0.1 to 1 v / v%, or 0.1 to 0.5 v / v%.

[0047] The pH of the cryopreservation solution according to the present invention may be adjusted as necessary. For example, if the hydrophilic group of the water-soluble polymer is a carboxyl group or the like, the cryopreservation solution containing such a water-soluble polymer may be acidic. In such cases, the cryopreservation solution can be made neutral by adjusting the pH, thereby making it more suitable for cryopreservation of biological samples. The type of salt used for pH adjustment is not particularly limited and can be appropriately selected from those commonly used for adjusting the pH of aqueous solutions.

[0048] (Physical Properties) In differential scanning calorimetry, the cryopreservation solution preferably exhibits an exothermic peak in the range of -30 to -10°C in a DSC curve obtained in the process of cooling from 20°C to -80°C. The heat of fusion calculated from the exothermic peak is preferably 250 J / g or less, and more preferably 0.1 J / g or more and 250 J / g or less. When the heat of fusion is 250 J / g or less, ice crystal formation during freezing can be reduced, and damage to cells during freezing can be reduced.

[0049] The exothermic peak temperature and heat of fusion of the cryopreservation solution can be measured using a differential scanning calorimeter (DSC) (e.g., Discovery DSC2500 manufactured by TA Instruments) under the following temperature conditions: First, the temperature is lowered from 20°C to -80°C at a cooling rate of 5°C / min, and then the solution is held at -80°C for 5 minutes, after which the temperature is raised to 20°C at a heating rate of 10°C / min. Then, from the obtained DSC curve, the temperature (°C) of the exothermic peak in the range of -30 to -10°C during the temperature lowering process is read, and the heat of fusion (J / g) is calculated.

[0050] The heat of fusion of the cryopreservation solution can be adjusted, for example, by the concentration or molecular weight of the water-soluble polymer in the cryopreservation solution, the total concentration of the polyhydric alcohol, etc. For example, the heat of fusion decreases as the molecular weight of the water-soluble polymer increases and the concentration of the water-soluble polymer or the total concentration of the polyhydric alcohol in the cryopreservation solution increases.

[0051] [Method for Cryopreserving Biological Samples] The method for freezing a biological sample using the cryopreservation solution of the present invention is not particularly limited. For example, the biological sample may be immersed in the cryopreservation solution of the present invention, and then the cryopreservation solution containing the biological sample may be frozen.

[0052] As described above, the type of biological tissue to be cryopreserved is not particularly limited. The cryopreservation solution of the present invention can cryopreserve biological samples such as cells and tissues with a high survival rate. Because the cryopreservation solution of the present invention is a non-permeable cryopreservation solution, it can be used to cryopreserve various types of cells. The species of organisms is also not particularly limited. Because the cryopreservation solution of the present invention can effectively suppress ice crystal formation and recrystallization during freezing and thawing, it can be suitably used to cryopreserve mammalian cells with complex structures. Examples of mammals include humans, mice, rats, cows, pigs, and dogs. Furthermore, the cryopreservation solution of the present invention can be suitably used to cryopreserve stem cells, early embryos, eggs, sperm, fertilized eggs, organized cell structures, tissues, and tissue-like materials (membranous materials or aggregates containing multiple cells), which are considered more difficult to cryopreserve than general cultured cells. Because the cryopreservation solution of the present invention does not require the use of differentiation-inducing agents such as DMSO or ethylene glycol, it can be used to preserve cells that require maintenance in an undifferentiated state. For example, stem cells for regenerative medicine can be cryopreserved without differentiation.

[0053] That is, the cryopreservation solution of the present invention can be used for cryopreserving somatic stem cells such as mesenchymal stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells, and germline stem cells, whether primary cells or established cells; blood cells; endothelial cells; etc. Furthermore, the cryopreservation solution of the present invention can be advantageously used for cryopreserving stem cells of primates, which are considered to have lower freeze tolerance than mice, for cryopreserving tissues for transplantation, and for cryopreserving germ cells in reproductive medicine.

[0054] The cryopreservation temperature of a biological sample is not particularly limited as long as the biological sample can be appropriately cryopreserved. For example, the cryopreservation temperature of a biological sample is −70° C. or lower, preferably −80° C. or lower. Furthermore, the cryopreservation temperature of a biological sample is −196° C. or higher, particularly preferably −150° C. or higher.

[0055] The cryopreservation solution according to the present invention is a cryopreservation solution for use in a slow freezing method. Therefore, in a freezing method using the cryopreservation solution according to the present invention, from the viewpoint of properly freezing a biological sample, the cooling rate is preferably 10°C / min or less, more preferably 5°C / min or less, and particularly preferably about 1°C / min.

[0056] By using the cryopreservation solution of the present invention, the vitrification state of the solvent is stabilized in the frozen state, and the toxicity of the cryopreservation solution itself is low, allowing biological samples to be stably preserved in the cryopreservation solution for long periods of time. As used herein, "long-term stable storage" means, for example, that the cell viability in a biological sample cryopreserved using the cryopreservation solution of the present invention is 90% or more (preferably 5% or more) after 5 months, 80% or more (preferably 90% or more) after 6 months, or 70% or more (preferably 85% or more) after 12 months, based on the cell viability immediately before storage. Furthermore, as used herein, "long-term stable storage" means, for example, that when a biological sample is frozen using the cryopreservation solution of the present invention and stored at -80°C for a long period of time, the biological sample is thawed, and the thawed biological sample is subsequently stored at 4°C, the cell viability in the biological sample stored at 4°C is 95% or more, based on the cell viability immediately after thawing, even 24 hours after thawing.

[0057] [Effects] The cryopreservation solution of the present invention contains a water-soluble polymer that can suppress cell rupture due to ice crystal formation by trapping water molecules during freezing and preventing ice crystal formation in the solvent while vitrifying the solution, and a cryoprotective support substance that can form ice nuclei in the extracellular solution at temperatures higher than the freezing point of intracellular water. Therefore, the cryopreservation solution of the present invention effectively suppresses ice crystal formation within and around cells and recrystallization upon thawing, thereby significantly reducing damage to cells during freezing and thawing. As a result, the cryopreservation solution of the present invention can cryopreserve various cells stably and with high viability, even without containing DMSO. Furthermore, the cryopreservation solution of the present invention has a viscosity of 30 mPa·s or less, which makes it less likely to foam during pipetting and provides excellent operability.

[0058] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0059] 1. Preparation of cryopreservation solutions (1) Cryopreservation solution 1 (MEM α medium containing 10% DMSO) Dimethyl sulfoxide (DMSO) (Nacalai Tesque, Inc.) was added to MEM α medium (Gibco) containing 10% fetal calf serum (FCS) and 1% penicillin / streptomycin to a final concentration of 10 w / v % to obtain a solution, which was designated as cryopreservation solution 1.

[0060] (2) Cryopreservation Solution 2 (Commercially Available DMSO-Free Cryopreservation Solution) A commercially available cell cryopreservation solution containing no serum or DMSO (STEM-CELLBANKER DMSO Free GMP grade, Nippon Zenyaku Kogyo Co., Ltd.) was used as cryopreservation solution 2.

[0061] (3) Cryopreservation Solution 3 (HBSS containing 5% hyaluronic acid) A solution obtained by adding low molecular weight hyaluronic acid to HBSS (Gibco) to a final concentration of 5 w / v% was used as cryopreservation solution 3.

[0062] (4) Cryopreservation solution 4 (HBSS containing 5% hyaluronic acid and 0.01% taurine) A solution obtained by adding low molecular weight hyaluronic acid to HBSS to a final concentration of 5 w / v% and taurine (Fujifilm Wako Pure Chemical Industries, Ltd.) to a final concentration of 0.01 w / v% was designated as cryopreservation solution 4.

[0063] (5) Cryopreservation solution 5 (HBSS containing 5% hyaluronic acid and 0.1% taurine) A solution obtained by adding low molecular weight hyaluronic acid to HBSS to a final concentration of 5 w / v% and taurine to a final concentration of 0.1 w / v% was designated as cryopreservation solution 5.

[0064] (6) Cryopreservation solution 6 (HBSS containing 5% hyaluronic acid and 1% taurine) A solution obtained by adding low molecular weight hyaluronic acid to HBSS to a final concentration of 5 w / v% and taurine to a final concentration of 1 w / v% was designated as cryopreservation solution 6.

[0065] (7) Cryopreservation solution 7 (HBSS containing 5% hyaluronic acid and 3% taurine) A solution obtained by adding low molecular weight hyaluronic acid to HBSS to a final concentration of 5 w / v% and taurine to a final concentration of 3 w / v% was designated as cryopreservation solution 7.

[0066] (8) Cryopreservation solution 8 (HBSS containing 5% hyaluronic acid and 5% taurine) A solution obtained by adding low molecular weight hyaluronic acid to HBSS to a final concentration of 5 w / v% and taurine to a final concentration of 5 w / v% was designated as cryopreservation solution 8.

[0067] (9) Cryopreservation solution 9 (HBSS containing 5% hyaluronic acid and 1% proline) A solution was obtained by adding low molecular weight hyaluronic acid to HBSS to a final concentration of 5 w / v% and proline (Fujifilm Wako Pure Chemical Industries, Ltd.) to a final concentration of 1 w / v%, and the resulting solution was designated as cryopreservation solution 9.

[0068] (10) Cryopreservation solution 10 (HBSS containing 5% hyaluronic acid and 3% proline) A solution obtained by adding low molecular weight hyaluronic acid to HBSS to a final concentration of 5 w / v% and proline to a final concentration of 3 w / v% was designated as cryopreservation solution 10.

[0069] (11) Cryopreservation solution 11 (HBSS containing 5% hyaluronic acid and 5% proline) A solution obtained by adding low molecular weight hyaluronic acid to HBSS to a final concentration of 5 w / v% and proline to a final concentration of 5 w / v% was used as cryopreservation solution 11.

[0070] (12) Cryopreservation solution 12 (HBSS containing 5% hyaluronic acid and 10% proline) A solution obtained by adding low molecular weight hyaluronic acid to HBSS to a final concentration of 5 w / v% and proline to a final concentration of 10 w / v% was used as cryopreservation solution 12.

[0071] (13) Cryopreservation solution 13 (HBSS containing 5% hyaluronic acid, 0.1% taurine, and 10% proline) A solution obtained by adding low-molecular-weight hyaluronic acid to HBSS to a final concentration of 5 w / v%, taurine to a final concentration of 0.1 w / v%, and proline to a final concentration of 10 w / v% was used as cryopreservation solution 13.

[0072] (14) Cryopreservation solution 14 (HBSS containing 5% hyaluronic acid, 0.1% taurine, 10% proline, and 10% glycerol) A solution obtained by adding low molecular weight hyaluronic acid to HBSS to a final concentration of 5 w / v%, taurine to a final concentration of 0.1 w / v%, proline to a final concentration of 10 w / v%, and glycerol (Fujifilm Wako Pure Chemical Industries, Ltd.) to a final concentration of 10 v / v% was used as cryopreservation solution 14.

[0073] (15) Cryopreservation solution 15 (HBSS containing 1% hyaluronic acid, 0.1% taurine, and 10% proline) A solution obtained by adding low-molecular-weight hyaluronic acid to HBSS to a final concentration of 1 w / v%, taurine to a final concentration of 0.1 w / v%, and proline to a final concentration of 10 w / v% was designated as cryopreservation solution 15.

[0074] (16) Cryopreservation solution 16 (oral rehydration solution containing 5% hyaluronic acid, 0.1% taurine, 10% proline, and 10% glycerol) An edible solution was obtained by adding low-molecular-weight hyaluronic acid to an oral rehydration solution (Aquarius Oral Rehydration Solution, Coca-Cola Japan Co., Ltd.) to a final concentration of 1 w / v%, taurine to a final concentration of 0.1 w / v%, proline to a final concentration of 10 w / v%, and glycerol to a final concentration of 10 v / v%, and the resulting solution was designated as cryopreservation solution 16.

[0075] (17) Cryopreservation Solution 17 (HBSS containing 10% hyaluronic acid) A solution obtained by adding low-molecular-weight hyaluronic acid to HBSS to a final concentration of 10 w / v % was used as cryopreservation solution 17.

[0076] 2. Measurement of viscosity-average molecular weight of water-soluble polymer The viscosity-average molecular weight of the low-molecular-weight hyaluronic acid used in preparing the above-mentioned cryopreservation solutions 3 to 17 was measured by the following procedure and found to be 13,600.

[0077] Low molecular weight hyaluronic acid was dissolved in a 0.2M aqueous sodium chloride solution (standard solution) and filtered to prepare diluted solutions with concentrations of 1.40g / dL, 1.20g / dL, 1.00g / dL, 0.80g / dL, 0.60g / dL, and 0.40g / dL. The outflow time of each diluted solution was measured using a 0B-type Ubbelohde viscometer at a temperature of 30°C. The outflow time of the standard solution was also measured in the same manner. The reduced viscosity I (dL / g) of each diluted solution was calculated using the following formula 3. In formula 3, t is the outflow time (seconds) of the diluted solution, and t ０ is the elution time of the standard solution (seconds), and c is the concentration of the diluent (g / dL).

[0078] The relationship between the concentration of each dilution and the reduced viscosity of the dilution (g / dL) was plotted on the horizontal axis and the reduced viscosity of the dilution (dL / g) on ​​the vertical axis, as shown in Figure 1. The intrinsic viscosity η (dL / g) was calculated from the intercept of the approximate line and was found to be 0.60 dL / g.

[0079] Viscosity average molecular weight M was calculated by the following formula 4, and was found to be 13,600. In formula 4, η is the intrinsic viscosity (dL / g). Table 1 shows the concentration of hyaluronic acid in the diluted solution, the reduced viscosity of each diluted solution, the intrinsic viscosity of hyaluronic acid, and the viscosity average molecular weight of hyaluronic acid.

[0080]

[0081] The steady flow viscosity of the prepared cryopreservation solutions was measured using a rheometer (MCR302, Anton Paar). The measurement conditions are as shown in Table 2. For comparison, the steady flow viscosities of HBSS, HBSS containing 5% hyaluronic acid and 10% glycerol (5% HA 10% Glyc), and HBSS containing 5% hyaluronic acid, 3% proline, and 5% glycerol (5% HA 3% Pro 5% Glyc) were also measured.

[0082]

[0083] The measurement results of some of the cryopreservation solutions and comparative solutions are shown in Figure 2 and Table 3. The cryopreservation solutions 4, 5, and 12 to 16, which are not shown in Figure 2 or Table 3, were also cryopreserved at 23°C for 100 seconds. －１ The viscosity of cryopreservation solutions 1 to 16 at a shear rate of 100 s was 30 mPa·s or less (20 mPa·s or less). Cryopreservation solutions 3 to 16 were less likely to foam when pipetted, making them easy to handle. －１ The viscosity of the cryopreservation solution 17 at a shear rate of 1000 kJ / min was more than 30 mPa·s (more than 40 mPa·s). The cryopreservation solution 17 was prone to foaming when pipetted, making it difficult to handle.

[0084] Viscosity η remained at a roughly constant value for 10s －１ ~1000s －１ When comparing the hyaluronic acid (HA) concentration and the viscosity, the viscosity increased significantly with increasing hyaluronic acid (HA) concentration. The addition of glycerol (Glyc) also increased the viscosity by approximately 10%. On the other hand, the addition of proline (Pro) and / or taurine (Tau) did not significantly affect the viscosity.

[0085] 4. Measurement of Heat of Fusion A portion of the prepared cryopreservation solution was subjected to DSC measurement under the following conditions using a differential scanning calorimeter (Discovery DSC2500, manufactured by TA Instruments). First, the temperature was lowered from 20°C to -80°C at a cooling rate of 5°C / min, and then the solution was held at -80°C for 5 minutes, after which the temperature was raised to 20°C at a heating rate of 10°C / min. The temperature (°C) of the exothermic peak in the temperature-lowering process in the range of -30 to -10°C was read from the obtained DSC curve, and the heat of fusion (J / g) was calculated. These results are shown in Table 3. In Table 3, values ​​not measured are indicated by "-". Cryopreservation solution 11, HBSS solution containing 5% hyaluronic acid and 10% glycerol, and HBSS solution containing 5% hyaluronic acid, 3% proline, and 5% glycerol all showed exothermic peaks in the range of -30 to -10°C, and the heat of fusion was 250 J / g or less.

[0086]

[0087] 5. Evaluation of Cryopreservation Solutions Cell freezing and thawing tests were carried out using the prepared cryopreservation solutions, and the cryopreservation solutions were evaluated based on the state of the thawed cells.

[0088] (Cell Culture) Human mesenchymal stem cells (ASC52telo, ATCC) were cultured in MEMα medium (Gibco) containing 10% FCS and 1% penicillin / streptomycin. When the cells reached approximately 80% confluence, they were harvested. Specifically, the cells were washed with PBS and detached using a trypsin solution (2.5 g / L trypsin, 1 mmol / L EDTA solution) (Nacalai Tesque, Inc.). The activity of the trypsin solution was suppressed by adding an equal or greater volume of medium. The solution was removed by centrifugation, and the cells were harvested.

[0089] (Freezing of cells) The collected cells were placed in the above-mentioned freezing medium at 1 × 10 ６ The cells were suspended at a concentration of 1000 cells / mL. The suspension was transferred into a screw-cap cryotube and used as a sample for freezing. Three samples were prepared for each type of cryopreservation solution. The cryopreservation container (Mr. Frosty, Nalgene) containing the sample was placed in a deep freezer at -80°C, and the sample was frozen at a rate of 1°C / min. The sample was then stored in the deep freezer at -80°C until a cell viability test was performed.

[0090] (Post-thaw cell viability assay) Frozen samples were thawed in a 37°C water bath, and the viability of the cells was assessed by flow cytometry. Specifically, cells were suspended in Flow Cytometry Staining Buffer (Invitrogen) and stained with propidium iodide (PI) (BioLegend) and fluorescent dye-labeled Annexin V (Invitrogen). For flow cytometry analysis, target cells were selected based on side-scattered light (SSC) and forward-scattered light (FSC). Target cells were classified into viable cells with minimal damage, viable cells with damage (potentially damaged cells), and dead cells based on the fluorescence intensity of PI (an indicator of cell membrane damage). Target cells were also classified into non-apoptotic and apoptotic cells based on the fluorescence intensity of fluorochrome-labeled Annexin V (an indicator of apoptosis).

[0091] 3A is a graph showing the ratio of live cells to dead cells after thawing for each cryopreservation solution. As shown in FIG. 3A, cryopreservation solutions 4 to 15 according to the present invention had a higher ratio of live cells than cryopreservation solution 1 containing DMSO. Furthermore, cryopreservation solutions 4 to 15 according to the present invention also had a higher ratio of live cells than cryopreservation solution 17, which corresponds to the cryopreservation solution described in Patent Document 1.

[0092] Figure 3B is a graph showing the proportion of intact live cells, damaged live cells, and dead cells after thawing for each cryopreservation solution. As shown in Figure 3B, when taurine was added to the cryopreservation solution (cryopreservation solutions 4-8), the proportion of damaged live cells was lowest when the taurine concentration was 1 w / v% (cryopreservation solution 6). Similarly, when proline was added to the cell cryopreservation solution (cryopreservation solutions 9-12), the proportion of damaged live cells was particularly low when the proline concentration was 5 w / v% or higher (cryopreservation solutions 11 and 12).

[0093] As shown in Table 4 below, when cryopreservation solution 1 containing DMSO was used, approximately 13.4% of cells were Annexin V staining-positive. This figure was nearly consistent with the percentage of dead cells detected by PI staining (14.3%). These results suggest that apoptosis is induced when cryopreservation solution 1 containing DMSO is used. On the other hand, when a cryopreservation solution without DMSO was used, no Annexin V staining-positive cells were detected. This indicates that the risk of apoptosis induction is low when a cryopreservation solution without DMSO is used.

[0094]

[0095] This application claims priority from Japanese Patent Application No. 2024-043638, filed March 19, 2024. The contents of the specification and drawings of that application are incorporated herein by reference in their entirety.

[0096] The cryopreservation solution and cryopreservation method according to the present invention are useful for cryopreserving biological samples such as cells and tissues in various fields such as regenerative medicine, livestock farming, and food.

Claims

1. A method for producing a solution containing an aqueous solvent, a water-soluble polymer, and at least one amino acid selected from the group consisting of glycine, alanine, valine, asparagine, isoleucine, glutamine, histidine, proline, and hydroxyproline, wherein the concentration of the amino acid is greater than 0.5 w / v%, and the solution is simmered at 23°C for 100 seconds. －１ A cryopreservation solution for biological samples, having a viscosity of 30 mPa·s or less at a shear rate of 1000 s.

2. A solution containing an aqueous solvent, a water-soluble polymer, and taurine, heated at 23°C for 100 seconds. －１ A cryopreservation solution for biological samples, having a viscosity of 30 mPa·s or less at a shear rate of 1000 s.

3. The cryopreservation solution for biological samples according to claim 1 or 2, wherein the viscosity is 5 mPa·s or more.

4. A cryopreservation solution for biological samples according to claim 1 or 2, in which, in differential scanning calorimetry, an exothermic peak is observed in the range of -30 to -10°C in a DSC curve obtained in the process of lowering the temperature from 20°C to -80°C, and the heat of fusion calculated from the exothermic peak is 250 J / g or less.

5. The cryopreservation solution according to claim 1 or 2, wherein the water-soluble polymer comprises at least one selected from the group consisting of hyaluronic acid and its salts, and alginic acid and its salts.

6. The cryopreservation solution according to claim 1, wherein the amino acid comprises proline.

7. The cryopreservation solution according to claim 6, wherein the concentration of the proline is within the range of 1 to 10 w / v%.

8. The cryopreservation solution according to claim 2, wherein the concentration of taurine is within the range of 0.01 to 5 w / v%.

9. The cryopreservation solution according to claim 2, further comprising proline.

10. The cryopreservation solution according to claim 1 or 2, wherein the concentration of dimethyl sulfoxide is 10 w / v% or less.

11. The cryopreservation solution according to claim 1 or 2, wherein the total concentration of the polyhydric alcohol selected from the group consisting of ethylene glycol, propylene glycol and glycerol is 30 v / v% or less.

12. The cryopreservation solution according to claim 1 or 2, wherein the biological sample is a cell.

13. A method for cryopreserving a biological sample, comprising the steps of: containing a biological sample in the cryopreservation solution according to claim 1 or 2; and freezing the cryopreservation solution containing the biological sample.

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