Cryopreservation solution for biological samples and method for cryopreserving biological samples

Abstract

The present invention provides a cryopreservation solution for biological samples that can cryopreserve biological samples with a survival rate equivalent to or better than that of DMSO, and that can significantly reduce the inclusion of air bubbles. [Solution] The cryopreservation solution comprises an aqueous solvent, at least one of hyaluronic acid and its salts having a weight-average molecular weight of 400 to 8800, and at least one amino acid selected from the group consisting of glycine, alanine, valine, asparagine, isoleucine, glutamine, histidine, proline, hydroxyproline, and taurine.

Description

[Technical Field]

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

[0002] Cryopreservation of biological samples such as cells and tissues is an important technique not only in the field of regenerative medicine but also in various fields such as livestock industry and food industry. 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 expanded in large quantities before use. In this case, it is common practice to cryopreserve the excess expanded cells, and thaw them for use when needed.

[0003] In cryopreservation of cells, when ice crystals grow inside cells, cell membranes and intracellular structures may be damaged, or cellular proteins may be denatured, resulting in fatal damage to the cells. To prevent such cell damage, slow freezing method, vitrification method and the like have been employed as methods for cryopreserving cells. In the slow freezing method, cells in a solution containing a low-concentration cryoprotectant are cooled slowly, so that the inside of the cells is dehydrated and frozen while the formation of intracellular ice crystals is prevented. In the vitrification method, cells in a solution containing a high-concentration cryoprotectant are rapidly cooled, thereby preventing the formation of ice crystals inside and outside the cells.

[0004] As a cryoprotectant, dimethyl sulfoxide (DMSO) is widely used. However, DMSO is known to have properties that adversely affect cells, particularly stem cells, such as cytotoxicity and differentiation-inducing activity. For this reason, development of cryopreservation solutions with low DMSO concentration or no DMSO has been promoted, and various solutions have been proposed (see, for example, Patent Document 1).

[0005] Patent Document 1 describes a cryopreservation solution comprising at least one selected from the group consisting of taurine, glycine, and their derivatives, a cryoprotective agent, a water-soluble polysaccharide (e.g., dextran), and an oligosaccharide (e.g., trehalose). This document states that the cryopreservation solution can suppress the decrease in cell viability even without containing DMSO. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2021-27 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] However, the cryopreservation solution described in Patent Document 1 has high viscosity, which makes it prone to foaming during pipetting and can lead to the entrapment of air bubbles, indicating room for improvement in terms of operability.

[0008] Therefore, an object of the present invention is to provide a cryopreservation solution for biological samples that can cryopreserve biological samples with an equivalent or better survival rate even without containing DMSO, and that can significantly reduce the entrapment of air bubbles. Another object of the present invention is to provide a method for cryopreserving biological samples using the above cryopreservation solution. [Means for solving the problem]

[0009] The present invention relates to the following cryopreservation solutions for biological samples and methods for cryopreserving biological samples. [1] A cryopreservation solution for biological samples comprising an aqueous solvent, at least one of hyaluronic acid and its salts having a weight-average molecular weight of 400 to 3000, and at least one amino acid selected from the group consisting of glycine, alanine, valine, asparagine, isoleucine, glutamine, histidine, proline, hydroxyproline, and taurine. [2] The cryopreservation solution according to [1], wherein the average value of the absorbance at a wavelength of 450 nm in the visible light absorption spectrum is 0.05 or less. [3] The cryopreservation solution according to [1] or [2], wherein the total concentration of at least one of the hyaluronic acid and its salts is 5 to 60 w / v%. [4] The cryopreservation solution according to any one of [1] to [3], wherein the molecular weight distribution of at least one of the hyaluronic acid and its salts is 1 to 4. [5] A cryopreservation solution according to any one of [1] to [4], further comprising hyaluronic acid having a weight-average molecular weight greater than 8800 and at least one of a salt thereof. [6] The cryopreservation solution according to [5], wherein the total concentration of hyaluronic acid and its salts having a weight-average molecular weight of 400 to 8800 is greater than the total concentration of hyaluronic acid and its salts having a weight-average molecular weight of more than 8800. [7] A cryopreservation solution according to any one of [1] to [6], comprising the proline. [8] The cryopreservation solution according to [7], wherein the concentration of proline is 1 to 7.5 w / v%. [9] A cryopreservation solution according to any one of [1] to [8], comprising the taurine.

[10] The cryopreservation solution according to [9], wherein the concentration of taurine is 0.01 to 5 w / v%.

[11] A cryopreservation solution according to any of [1] to

[10] , wherein the concentration of dimethyl sulfoxide is 3 w / v% or less.

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

[11] , wherein the total concentration of polyhydric alcohols selected from the group consisting of ethylene glycol, propylene glycol, and glycerol is 0.1 to 15 v / v%.

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

[12] , wherein the total concentration of at least one of the hyaluronic acid and its salts is 10 to 60 w / v%.

[14] The biological sample is a cell, and the cryopreservation solution is one of the following: [1] to

[13] . A method for cryopreserving a biological sample, comprising the steps of: including a biological sample in a cryopreservation solution described in any of

[15] [1] to

[14] ; and freezing the cryopreservation solution containing the biological sample. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide a cryopreservation solution for biological samples that can cryopreserve biological samples with a survival rate equivalent to or better than that of DMSO, and that can significantly reduce the entrapment of air bubbles. Furthermore, according to the present invention, it is possible to provide a method for cryopreserving biological samples using the above-mentioned cryopreservation solution. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 is a photograph showing the results of the defoaming test. [Figure 2] Figure 2 shows phase-contrast images of cells that were thawed in each cryopreservation solution and then seeded in cell culture vessels.

[0012] The embodiments of the present invention will be described in detail below. However, the present invention is not limited to these embodiments.

[0013] In this specification, a numerical range represented by "~" means a range that includes the numbers before and after "~" as the lower and upper limits. In numerical ranges described stepwise in this specification, the upper or lower limit described in one numerical range may be replaced by the upper or lower limit of another numerical range described stepwise.

[0014] Our research has revealed that hyaluronic acid or its salts are water-soluble polymers that can trap water molecules during freezing, preventing ice crystal formation in the solvent portion while vitrifying, and thus suppressing cell rupture caused by ice crystal formation. The present inventors have found that by setting the molecular weight of hyaluronic acid or a salt thereof to a predetermined value or lower, the viscosity of a cryopreservation solution can be appropriately lowered, and foaming during pipetting can be highly suppressed. On the other hand, when the molecular weight of hyaluronic acid or a salt thereof is lowered, it is generally expected that it becomes difficult to obtain the effect of trapping water molecules during freezing and suppressing cell rupture caused by ice crystal formation. However, the present inventors have found that by combining hyaluronic acid or a salt thereof with a predetermined amino acid (a cryoprotection support substance), cell survival rate equivalent to or higher than that obtained with dimethyl sulfoxide can be achieved even when dimethyl sulfoxide is not contained. Hereinafter, a cell cryopreservation solution according to one embodiment of the present invention will be specifically described.

[0015] [Cell Cryopreservation Solution] A cryopreservation solution for biological samples (hereinafter also referred to as "cryopreservation solution") comprises an aqueous solvent, at least one of hyaluronic acid and a salt thereof having a weight average molecular weight of 400 to 8800 (hereinafter also referred to as "hyaluronic acids"), and a cryoprotection support substance. The cryoprotection 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 subjected to cryopreservation is not particularly limited as long as it contains cells. Examples of biological tissues include cells (e.g., stem cells, germ cells, somatic cells, fertilized eggs, etc.), early embryos, tissues, organized cell structures, and tissue-like materials (e.g., membrane-like materials and aggregates containing a large number of cells). The species of the organism from which the biological tissue is derived is also not particularly limited.

[0017] Hereinafter, each component will be specifically described.

[0018] (Aqueous Solvent) The type of aqueous solvent is not particularly limited as long as the biological sample can be properly cryopreserved. The aqueous solvent may be, for example, an isotonic solution whose salt concentration or sugar concentration has been adjusted with sodium ions, potassium ions, calcium ions, etc., so that it is approximately the same as the osmotic pressure of body fluids or cell fluid. Examples of aqueous solvents include water; physiological saline; buffered physiological salines such as phosphate-buffered saline (PBS), Dulbecco's phosphate-buffered saline, Tris-buffered saline (TBS), and HEPES-buffered saline; equilibrium salt solutions such as Hanks' equilibrium salt solution (HBSS); Ringer's solution such as Ringer's solution, lactated 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 also contain other optional components such as isotonic agents, chelating agents, solubilizers, pH adjusters, and additives commonly used as additives to cell culture media.

[0019] The aqueous solvent may be a commercially available culture medium or a cell culture medium such as D-MEM, E-MEM, αMEM, RPMI-1640 medium, Ham's F-12, Ham's F-10, or M-199. The cryopreservation solution may also include a cell culture or cell suspension to which hyaluronic acid or its salt and cryoprotection support substances have been added at a predetermined concentration.

[0020] (Hyaluronic acid derivatives) Hyaluronic acid derivatives are at least one of hyaluronic acid and its salts, having a weight-average molecular weight of 400 to 8800.

[0021] Hyaluronic acid is a sugar that contains one or more constituent units consisting of a disaccharide of glucuronic acid and N-acetylglucosamine. Hyaluronic acid or its salts may be extracted from natural products of animals, etc. (for example, living tissues such as chicken combs, umbilical cords, skin, and synovial fluid), or they may be synthesized chemically or enzymatically. Hyaluronic acid or its salts having a weight-average molecular weight of 400 to 8800 may be low molecular weight products (cleavage products or fragments of hyaluronic acid or its salts) obtained by decomposing high molecular weight hyaluronic acid or its salts, as described later, and may also be hyaluronic acid oligosaccharides (including disaccharides).

[0022] While not particularly limited, hyaluronic acid salts include metal salts and ammonium salts. Examples of metal salts include monovalent metal salts such as alkali metal salts, and divalent or more valent metal salts such as alkaline earth metal salts and zinc salts. Examples of hyaluronic acid salts include sodium salt, potassium salt, calcium salt, zinc salt, magnesium salt, and ammonium salt. These may be present individually or in combination of two or more types.

[0023] The weight-average molecular weight of hyaluronic acid or its salt is 400 to 8800, as described above. A weight-average molecular weight of hyaluronic acid or its salt of 8800 or less lowers the viscosity of the cryopreservation solution, effectively suppressing foaming during pipetting, etc. This improves the handling of the cryopreservation solution. On the other hand, a weight-average molecular weight of hyaluronic acid or its salt of 400 or more facilitates vitrification during freezing, thereby increasing cell viability. From a similar viewpoint, a weight-average molecular weight of hyaluronic acid or its salt of 500 to 8000 is more preferable, and 600 to 7500 is even more preferable. That is, hyaluronic acid or its salt is preferably hyaluronic acid oligosaccharide or its salt. In other embodiments, the weight-average molecular weight of hyaluronic acid or its salt may be 600 to 1800. Hyaluronic acid or its salt with a weight-average molecular weight of 400 to 8800 may be included as one type or as two or more types. Furthermore, a weight-average molecular weight of 8800 in hyaluronic acid is roughly equivalent to a viscosity-average molecular weight of around 3000.

[0024] Furthermore, the molecular weight distribution (Mw / Mn) of at least one of hyaluronic acid and its salts is not particularly limited, but is preferably 1 to 10, and more preferably 1 to 4. When the molecular weight distribution of hyaluronic acid is 10 or less, the viscosity of the cryopreservation solution tends to be lower, and foaming during pipetting can be further suppressed.

[0025] The weight-average molecular weight and molecular weight distribution of hyaluronic acid or its salts can be measured by gel permument chromatography (GPC). Specifically, a solution is obtained by dissolving hyaluronic acid or a salt thereof in a 0.1 mol / L NaNO3 aqueous solution under the following conditions. GPC measurement is performed on this solution under the following conditions, and the resulting molecular weight distribution curve is calibrated using a polyethylene glycol / polyethylene oxide standard to determine the weight-average molecular weight (Mw) of hyaluronic acid or a salt thereof. (GPC measurement conditions) • Measurement equipment: GPC system (e.g., KP-22-13S dual pump (Fromm), automatic injection system 717plus (Waters), differential refractive index detector RI-101 (Shodex)) • Data analysis software: Empower3 (Waters Corporation) • Detector: Differential refractometer (RI) • Series-linked columns: Shodex OHpak 13μ SB-806M HQ (8.0mm x 30mm) x 2 (manufactured by Resonaq) Column temperature: 40°C ·Flow rate: 1.0mL / min • Sample concentration: 0.1 w / v% ·Injection volume: 100μL

[0026] Hyaluronic acid or its salts having the weight-average molecular weight described above, such as hyaluronic acid oligosaccharides or their salts, can be obtained by enzymatic decomposition or hydrolysis with acid or alkali from high molecular weight hyaluronic acid or its salts. The manufacturing method is not limited to these. Since a lower molecular weight of hyaluronic acid or its salts tends to result in a lower viscosity of the cryopreservation solution, the desired viscosity range can be obtained by adjusting the degree of decomposition of the hyaluronic acid or its salts.

[0027] The hyaluronic acid or its salts described above have a molecular weight above a certain level and a large number of hydrophilic groups. When a biological sample is cryopreserved using a cryopreservation solution containing such hyaluronic acid or its salts, during the cooling process of the biological sample being cryopreserved, water molecules of the solvent are trapped in the matrix formed by the hyaluronic acid or its salts. At this time, because the molecular chains contain hydrophilic groups, the molecular motion of water is restricted during cooling, and the water can solidify and / or freeze in a vitrified state without crystallizing. Due to the action of these molecular chains, the inside of the cell is also dehydrated and vitrified, so when cryopreserving using a cryopreservation solution, it is not necessary to increase the concentration of the solute (cryoprotectant) or increase the cooling rate, as is done in conventional vitrification methods. In this embodiment, since the formation of ice crystals inside the cell is suppressed by the action of the molecular chains, the osmotic shock to the cell during freezing, which was a problem in conventional vitrification methods that use the osmotic pressure difference between the inside and outside of the cell to dehydrate the inside of the cell and vitrify the inside of the cell, can be weakened. In addition, since recrystallization does not occur when the frozen cell is thawed, it is thought that there is less damage to the cell due to thawing.

[0028] The total concentration of hyaluronic acid and its salts in the cryopreservation solution is not particularly limited, but is preferably 1 to 60 w / v%, more preferably 2 to 50 w / v%, even more preferably 3 to 30 w / v%, and particularly preferably 5 to 15 w / v%. If the total concentration of hyaluronic acid and its salts is 60 w / v% or less, the viscosity of the cryopreservation solution can be made more appropriately low, which can suppress foaming during pipetting, for example, and improve operability. If the total concentration of hyaluronic acid and its salts is 1 w / v% or more, during the cooling process of the biological sample, water molecules of the solvent are more easily trapped in the matrix formed by the hyaluronic acid or its salts, making it easier to vitrify the water without crystallizing it. Therefore, it is possible to make it more difficult for ice crystals to form outside the cells and to better maintain cell viability.

[0029] (Freeze protection support substance) Cryoprotection support substances are substances that can form ice nuclei in the extracellular solution at a temperature higher than the freezing temperature of intracellular water. Cryopreservation solutions have a low molecular weight of hyaluronic acid or its salts to minimize foaming. However, a lower molecular weight of hyaluronic acid or its salts makes it more difficult to trap water molecules and vitrify during freezing compared to a higher molecular weight, which can be detrimental to cell viability. Even in such cases, adding further cryoprotection support substances can help maintain good cell viability.

[0030] As mentioned above, the cryoprotection support substance contains at least one amino acid selected from the group consisting of glycine, alanine, valine, asparagine, isoleucine, glutamine, histidine, proline, hydroxyproline, and taurine. For example, the cryoprotection 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. Alternatively, the cryoprotection support substance may be taurine. The cryopreservation solution may contain only one of the above compounds, or it may contain two or more. For example, the cryopreservation solution may contain both the above amino acid (e.g., proline) and taurine. This effectively enhances the cell-protective function during freezing, thereby increasing the cell viability.

[0031] The concentration of the cryoprotection support substance is not particularly limited as long as the effect of the cryoprotection support substance can be appropriately exerted. For example, if the cryoprotection support substance is an amino acid other than taurine (e.g., proline), the concentration of the amino acid is preferably greater than 0.5 w / v%, more preferably 1 to 7.5 w / v%, even more preferably 3 to 7.5 w / v%, and particularly preferably 5 to 7.5 w / v%. If the cryoprotection support substance contains multiple types of amino acids, the above concentration refers to the total concentration of the amino acids. Furthermore, the ratio of the concentration of the amino acid other than taurine (w / v%) to the concentration of hyaluronic acid or its salt (w / v%) (amino acid other than taurine / hyaluronic acid or its salt) is preferably 0.06 to 3, more preferably 0.1 to 2, and particularly preferably 0.1 to 1.

[0032] On the other hand, when the cryoprotection support substance is taurine, the concentration of taurine is not particularly limited, but is preferably 0.01 to 5 w / v%, more preferably 0.5 to 2 w / v%, and particularly preferably about 1 w / v%. Furthermore, the ratio of the concentration of taurine (w / v%) to the concentration of hyaluronic acid or its salt (w / v%) (taurine / hyaluronic acid or its salt) is preferably, for example, 0.01 to 0.3, and more preferably 0.02 to 0.2.

[0033] When taurine and other amino acids (e.g., proline) are used in combination as cryoprotection support substances, the ratio of the concentration of taurine (w / v%) to the concentration of the other amino acids (w / v%) (taurine / other amino acids) is preferably 0.0006 to 1, and more preferably 0.1 to 0.2.

[0034] (Other ingredients) The cryopreservation solution may further contain other optional components, as long as they do not impair the effects of the present invention.

[0035] For example, the cryopreservation solution may further contain hyaluronic acid or a salt thereof having a weight-average molecular weight greater than 8800 (hereinafter collectively referred to as "second hyaluronic acid derivatives").

[0036] In other words, hyaluronic acid or its salts with a polymerization average molecular weight of 400 to 8800, as described above, can lower the viscosity of the cryopreservation solution, but there is room for improvement in cell viability. In contrast, by further including hyaluronic acid or its salts with a higher molecular weight than those described above, it is possible to suppress foaming to a high degree while further increasing cell viability.

[0037] Hyaluronic acid or its salts with a weight-average molecular weight exceeding 8800 are more likely to trap moisture and vitrify during freezing, thereby increasing cell viability. The weight-average molecular weight of the hyaluronic acid or its salt is preferably between 8800 and 270000, more preferably between 10000 and 135000, and even more preferably between 10000 and 40000. When the weight-average molecular weight of the hyaluronic acid or its salt is 270000 or less, the viscosity of the cryopreservation solution can be further reduced, and foaming can be suppressed even more effectively.

[0038] If the cryopreservation solution further contains hyaluronic acid and its salts having a weight-average molecular weight of over 8800, it is preferable that the total concentration of the hyaluronic acid and its salts having a weight-average molecular weight of 400 to 8800 is higher than the total concentration of the hyaluronic acid and its salts having a weight-average molecular weight of over 8800. Specifically, the mass ratio of the total concentration of the above hyaluronic acid compounds to the total concentration of the second hyaluronic acid compound in the cryopreservation solution is preferably, for example, 100:1 to 1.1:1, and more preferably 20:1 to 1.5:1.

[0039] The total concentration of hyaluronic acid and its salts with a weight-average molecular weight exceeding 8800 in the cryopreservation solution is not particularly limited as long as it does not impair the effects of the present invention, but is preferably 0 to 10 w / v%, and more preferably 0.1 to 5 w / v%. If the total concentration of hyaluronic acid and its salts with a weight-average molecular weight exceeding 8800 is 10 w / v% or less, the viscosity of the cryopreservation solution can be made less likely to increase, thus further suppressing foaming during pipetting and improving operability. If the total concentration of hyaluronic acid and its salts with a weight-average molecular weight exceeding 8800 is 0.1 w / v% or more, water molecules can be more easily trapped by the matrix formed by the second hyaluronic acid during the cooling process of the biological sample, making it easier to vitrify water without crystallizing it. Therefore, it is less likely to form ice crystals outside the cell, and the cell viability can be further increased.

[0040] Furthermore, the cryopreservation solution may also contain polyhydric alcohols such as ethylene glycol, propylene glycol, and glycerol. Polyhydric alcohols can be used as cryoprotective agents to suppress the formation of ice crystals within cells. For example, even if the concentration of hyaluronic acid or its salts in the cryopreservation solution is low, the inclusion of polyhydric alcohols can further increase the viability of cells. The total concentration of polyhydric alcohols selected from the group consisting of ethylene glycol, propylene glycol, and glycerol in the cryopreservation solution is preferably 0 to 15 v / v%. From the viewpoint of further increasing cell viability, the total concentration of polyhydric alcohols in the cryopreservation solution is more preferably 0.1 to 15 v / v%, and even more preferably 3 to 10 v / v%. On the other hand, from the viewpoint of further improving the safety and operability of the cryopreservation solution, the total concentration of polyhydric alcohols is preferably low, more preferably 0 to 5 v / v%, even more preferably 0 to 1 v / v%, even more preferably 0 to 0.5 v / v%, and particularly preferably 0 to 0.1 v / v%.

[0041] Furthermore, the cryopreservation solution may contain cell membrane-impermeable cryoprotective agents such as sugars and dextran. Examples of sugars include dextrose, mannose, galactose, fructose, raffinose, lactose, sucrose, maltose, glucose, sorbitol, mannitol, and trehalose. The cryopreservation solution may contain such cell membrane-impermeable cryoprotective agents at a concentration of, for example, 0.1 to 10 w / v%.

[0042] 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 0-3 w / v%, more preferably 0-1 w / v%, even more preferably 0-0.5 w / v%, and particularly preferably 0-0.1 w / v%. By reducing the concentration of DMSO, problems such as cytotoxicity and differentiation-inducing properties of DMSO can be resolved. Furthermore, the concentration of dimethyl sulfoxide (DMSO) in the cryopreservation solution may be 0.1-10 w / v%, 0.1-5 w / v%, 0.1-1 w / v%, or 0.1-0.5 w / v%.

[0043] The pH of the cryopreservation solution may be adjusted as needed. The type of salt used for pH adjustment is not particularly limited and can be appropriately selected from those commonly used for pH adjustment of aqueous solutions.

[0044] (Physical properties) When cryopreservation solutions contain cytotoxic substances such as DMSO, a process is required to remove the cryopreservation solution by centrifugation, resuspend the cells in culture medium, and then seed them in culture vessels. On the other hand, the cryopreservation solution of this embodiment substantially does not contain cytotoxic components such as DMSO, so the cryopreservation solution can be directly diluted with culture medium and used to seed cells in culture vessels. This makes it promising for use in regenerative medicine, drug discovery screening that processes large quantities of cells, and safety testing. In this case, from the perspective of cell culture, it is preferable that the solution has little color and is nearly transparent. Conventional cryopreservation solutions containing DMSO have a strong yellowish tint. In contrast, the cryopreservation solution of this embodiment has less yellowish tint because, although it depends on the concentration of hyaluronic acid or its salt, the weight-average molecular weight of the hyaluronic acid or its salt is relatively low at 3000 or less. The culture medium contains phenol red as a pH indicator, which is red when neutral and unused, but changes to yellow when acidic. The coloring of the cryopreservation solution inhibits (makes less visible) this color change, so the cryopreservation solution of this embodiment, which has less yellowish tint, is more suitable for cell culture after freeze-thawing. Specifically, the average absorbance of the cryopreservation solution at a wavelength of 450 nm is preferably 0.05 or less, and more preferably between 0 and 0.048, although this also depends on the total concentration of hyaluronic acid and its salts.

[0045] The absorbance of the cryopreservation solution can be measured at a wavelength of 450 nm using a spectrophotometer (e.g., a plate reader (SPECTRA max PLUS384, manufactured by Molecular Devices)). Ten absorbance measurements should be taken, and the average value of these measurements can be obtained as the "average absorbance."

[0046] The absorbance of the cryopreservation solution can be adjusted, for example, by the molecular weight and concentration of hyaluronic acid and its salts, which have a weight-average molecular weight of 400 to 8800 as described above. If the molecular weight and concentration of hyaluronic acid and its salts in the cryopreservation solution are low, the absorbance of the cryopreservation solution tends to be low.

[0047] [Methods for cryopreserving biological samples] The method for freezing biological samples using cryopreservation solutions is not particularly limited. For example, the biological sample can be placed in the cryopreservation solution, and then the cryopreservation solution containing the biological sample can be frozen.

[0048] As mentioned above, the types of biological tissues that can be cryopreserved are not particularly limited. The cryopreservation solution can cryopreserve biological samples such as cells and tissues with a high viability rate. Because the cryopreservation solution is an impermeable cryopreservation solution, it can be used for the cryopreservation of various types of cells. The species of organism is also not particularly limited. Because the cryopreservation solution can effectively suppress ice crystal formation and recrystallization during freezing and thawing, it can be suitably used for the cryopreservation of mammalian cells with complex structures. Examples of mammals include humans, mice, rats, cattle, pigs, and dogs. Furthermore, the cryopreservation solution can be suitably used for the cryopreservation of stem cells, early embryos, eggs, sperm, fertilized eggs, organized cell structures, tissues, and tissue-like substances (membrane-like substances and aggregates containing many cells), which are considered more difficult to cryopreserve than general culture cells. Because the cryopreservation solution does not need to contain differentiation-inducing substances such as DMSO or ethylene glycol, it can be used to preserve cells that need to be kept in an undifferentiated state. For example, stem cells for regenerative medicine applications can be cryopreserved without differentiation.

[0049] In other words, cryopreservation solutions can be used for the cryopreservation of somatic stem cells such as mesenchymal stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells, and germ stem cells, as well as blood cells and endothelial cells, regardless of whether they are primary or established cells. Furthermore, cryopreservation solutions can be advantageously used for the cryopreservation of primate stem cells, which are considered to have lower cryopreservation tolerance than mice, for the cryopreservation of tissues for transplantation, and for the cryopreservation of germ cells in reproductive medicine.

[0050] The freezing temperature for biological samples is not particularly limited as long as the biological samples can be properly frozen and stored. For example, the freezing temperature for biological samples is -70°C or lower, and preferably -80°C or lower. Furthermore, the freezing temperature for biological samples is -196°C or higher, and particularly preferably -150°C or higher.

[0051] The cryopreservation solution is a cryopreservation solution for the slow freezing method. Therefore, in a freezing method using the cryopreservation solution, from the viewpoint of properly freezing the 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.

[0052] By using a cryopreservation solution, the vitrification state of the solvent is stabilized in the frozen state, and the cryopreservation solution itself has low toxicity, so that biological samples can be stored stably for a long period of time in the cryopreservation solution. In this specification, "stable storage for a long period of time" means, for example, that the viability of cells in a biological sample cryopreserved using a cryopreservation solution is 90% or more (preferably 5% or more) after 5 months, or 80% or more (preferably 90% or more) after 6 months, or 70% or more (preferably 85% or more) after 12 months, based on the viability of cells immediately before storage. In addition, in this specification, "stable storage for a long period of time" means, for example, that when a biological sample is frozen using a cryopreservation solution and stored at -80°C for a long period of time, then the biological sample is thawed, and subsequently stored at 4°C, the viability of cells in the biological sample stored at 4°C is 95% or more even 24 hours after thawing, based on the viability of cells immediately after thawing.

[0053] [effect] The cryopreservation solution contains hyaluronic acid or a salt thereof, which can suppress cell rupture due to ice crystal formation by trapping water molecules during freezing and vitrifying the solvent while preventing ice crystal formation, and a cryoprotection support substance that can form ice nuclei in the extracellular solution at a temperature higher than the freezing temperature of intracellular water. Therefore, the cryopreservation solution can suppress ice crystal formation inside and around cells and recrystallization during thawing, thereby reducing damage to cells during freezing and thawing. As a result, the cryopreservation solution can cryopreserve various cells while maintaining a viability rate above a predetermined level, even without containing DMSO. Furthermore, because the molecular weight of hyaluronic acid or its salt is within a predetermined range, the cryopreservation solution has a moderately low viscosity, resulting in very little foaming during pipetting and excellent handling. [Examples]

[0054] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0055] 1. Preparation of cryopreservation solution (1) Cryopreservation solution 1 (HBSS containing 5% low molecular weight hyaluronic acid, 5% proline, 1% taurine, and 10% glycerol) A solution obtained by adding low molecular weight hyaluronic acid (microHA, manufactured by Blue Mage Biotechnology, Mw / Mn=2.395~2.517, viscosity-average molecular weight 2300 (estimated calculation)) with a weight-average molecular weight of 6600 to HBSS at a final concentration of 5 w / v%, proline (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) at a final concentration of 5 w / v%, taurine (manufactured by Mitsui Chemicals, Inc.) at a final concentration of 1 w / v%, and glycerol at a final concentration of 10 v / v% was designated as cryopreservation solution 1.

[0056] (2) Cryopreservation solution 2 (HBSS containing 20% ​​low molecular weight hyaluronic acid, 5% proline, 1% taurine, and 10% glycerol) A solution was prepared by adding low molecular weight hyaluronic acid (microHA, manufactured by Blue Mage Biotechnology, with a weight-average molecular weight of 6600, Mw / Mn = 2.395~2.517, viscosity-average molecular weight of 2300 (estimated calculation)) to HBSS, changing the final concentration from 20 w / v% to 1 w / v%, adding proline (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to a final concentration of 5 w / v%, adding taurine (manufactured by Mitsui Chemicals, Inc.) to a final concentration of 1 w / v%, and adding glycerol to a final concentration of 10 v / v%, and this solution was designated as cryopreservation solution 2.

[0057] (3) Cryopreservation solution 3 (HBSS containing 30% low molecular weight hyaluronic acid, 5% proline, 1% taurine, and 10% glycerol) A solution obtained by adding low molecular weight hyaluronic acid (microHA, manufactured by Blue Mage Biotechnology, Mw / Mn=2.395~2.517, viscosity-average molecular weight 2300 (estimated calculation)) with a weight-average molecular weight of 6600 to HBSS at a final concentration of 30 w / v%, proline (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) at a final concentration of 5 w / v%, taurine (manufactured by Mitsui Chemicals, Inc.) at a final concentration of 1 w / v%, and glycerol at a final concentration of 10 v / v% was designated as cryopreservation solution 3.

[0058] (4) Cryopreservation solution 4 (HBSS containing 5% low molecular weight hyaluronic acid, 0.9% high molecular weight hyaluronic acid, 5% proline, 1% taurine, and 10% glycerol) A solution was prepared by adding low molecular weight hyaluronic acid (microHA, Mw / Mn=2.395~2.517, viscosity-average molecular weight 2300 (estimated calculation)) with a weight-average molecular weight of 6600 to HBSS at a final concentration of 5 w / v%, hyaluronic acid (Fuji Medicie Co., Ltd.) with a weight-average molecular weight of 40000 and a viscosity-average molecular weight of 13600 at a final concentration of 0.9 w / v%, proline (Fujifilm Wako Pure Chemical Industries, Ltd.) at a final concentration of 5 w / v%, taurine (Fujifilm Wako Pure Chemical Industries, Ltd.) at a final concentration of 1 w / v%, and glycerol at a final concentration of 10 v / v% to HBSS, and this was designated as cryopreservation solution 4.

[0059] (5) Cryopreservation solution 5 (HBSS containing 5% hyaluronic acid, 5% proline, 1% taurine, and 10% glycerol) A solution obtained by adding hyaluronic acid (manufactured by Fuji Medicie Co., Ltd.) with a weight-average molecular weight of 40,000 and a viscosity-average molecular weight of 13,600 to HBSS at a final concentration of 5 w / v%, proline (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) at a final concentration of 5 w / v%, taurine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) at a final concentration of 1 w / v%, and glycerol at a final concentration of 10 v / v% was designated as cryopreservation solution 5.

[0060] (6) Cryopreservation solution 6 (commercially available cryopreservation solution containing 10% DMSO) A commercially available cryopreservation solution containing dimethyl sulfoxide (DMSO) and no serum (Banbankar, manufactured by GC Lymphotec) was designated as cryopreservation solution 6.

[0061] 2. Measurement of the molecular weight of hyaluronic acid 2-1. Measurement of weight-average molecular weight The weight-average molecular weight (Mw) of the hyaluronic acid used in the preparation of the above cryopreservation solutions 1 to 5 was measured by gel permulation chromatography (GPC). Specifically, hyaluronic acid was dissolved in a 0.1 mol / L NaNO3 aqueous solution under the following conditions to obtain a solution. GPC measurements were performed on this solution under the following conditions, and the weight-average molecular weight (Mw) of hyaluronic acid was determined by calibrating the resulting molecular weight distribution curve using a polyethylene glycol / polyethylene oxide standard. (GPC measurement conditions) • Measuring equipment: KP-22-13S dual pump (Fromm), automatic injection device 717plus (Waters), differential refractive index detector RI-101 (Shodex) • Data analysis software: Empower3 (Waters Corporation) • Detector: Differential refractometer (RI) • Series-linked columns: Shodex OHpak 13μ SB-806M HQ, 8.0mm x 30mm (2 tubes) (manufactured by Resonaq) Column temperature: 40°C ·Flow rate: 1.0mL / min • Sample concentration: 0.1 w / v% ·Injection volume: 100μL

[0062] 2-2. Measurement of viscosity-average molecular weight The viscosity-average molecular weight of the hyaluronic acid (manufactured by Fuji Medicie Co., Ltd.) used in the preparation of the above cryopreservation solutions 4 and 5 was measured by the following method. Specifically, the above-mentioned hyaluronic acid was dissolved in a 0.2 M sodium chloride aqueous solution (standard solution), filtered, and diluted solutions were prepared with concentrations of 1.40 g / dL, 1.20 g / dL, 1.00 g / dL, 0.80 g / dL, 0.60 g / dL, and 0.40 g / dL, respectively. The elution time of each diluted solution was measured using an OB-type Ubbelohde viscometer at a temperature of 30°C. Similarly, the elution time of the standard solution was also measured. For each diluted solution, the reduced viscosity I (dL / g) was calculated using the following formula 1. In formula 1, t is the elution time of the diluted solution (seconds), t0 is the elution time of the standard solution (seconds), and c is the concentration of the diluted solution (g / dL).

number

[0063] The relationship between the concentration of the diluent (g / dL) and the reduced viscosity of the diluent (dL / g) was plotted on the x-axis and y-axis, respectively. The intrinsic viscosity η (dL / g) was calculated from the intercept of the approximation line and was found to be 0.60 dL / g.

[0064] The viscosity-average molecular weight M for the above hyaluronic acid was calculated using the following Equation 2, and it was found to be 13600. In Equation 2, η is the intrinsic viscosity (dL / g).

number

[0065] 3. Evaluation of cryopreservation solutions 3-1. Presence or absence of bubbles (visual inspection) 0.5 mL of the obtained cryopreservation solution was placed in a 1.5 mL Eppendorf tube, and the mixture was stirred for 1 minute using a vortex mixer (Vortex-Genie 2, Kenis) with the vibration intensity set to the line between 7 and 8 on the scale. After that, it was allowed to stand for 5 minutes. After that, the presence or absence of bubbles was checked visually. A: There were no bubbles. B: There wasn't enough foam. C: There was a lot of foam.

[0066] 3-2.Absorbance The absorbance of the obtained cryopreservation solution at a wavelength of 450 nm was measured using a plate reader (SPECTRA max PLUS384, Molecular Devices).

[0067] 3-3. Color (visual inspection) 0.5 mL of the obtained cryopreservation solution was placed in a 1.5 mL Eppendorf tube and allowed to stand, after which the color was visually evaluated. A: It was colorless and transparent. B: It had a slightly yellowish tint. C: It was very yellowish.

[0068] 3-4. Cell viability We performed a freeze-thaw test on cells using the prepared cryopreservation solution and evaluated the cell viability based on the state of the thawed cells.

[0069] (Cell culture) Human mesenchymal stem cells (hMSCs, PT-2501, LONZA) were cultured in MSCGM medium containing serum (PT-3001, LONZA). When the cells reached approximately 80% confluence, they were harvested. Specifically, the cells were washed with PBS, detached using trypsin / EDTA solution (CC-3232, LONZA), the activity of the trypsin solution was suppressed by adding an equal or greater volume of culture medium, the solution was removed by centrifugation, and the cells were harvested.

[0070] (Cell freezing) The recovered cells were placed in the above cryopreservation solution in a 1 × 10⁶ solution. 6 The cells were suspended at a concentration of cells / mL. The suspension was transferred to a screw-cap cryotube to be used as the freezing sample. Three samples were prepared for each type of cryopreservation solution. The samples were frozen at a rate of 1°C / min by placing the cryopreservation container (CoolCell, Corning) containing the samples into a -80°C deep freezer. After that, the samples were stored in the -80°C deep freezer for 24 hours until cell viability testing was performed, then removed from the cryopreservation container and transferred to a liquid nitrogen tank for 3 days.

[0071] (Cell viability test after thawing) Frozen samples were thawed in a 37°C water bath for 1 minute and 30 seconds, and the viability of the cells was determined using a hemocytometer. Specifically, cells were suspended in culture medium, then suspended in an equal volume of trypan blue solution (MP-Biomedicals), and 10 μL was added to the hemocytometer. Cells stained blue with trypan blue were considered dead cells, and the number of viable and dead cells was counted under a phase-contrast microscope (CKX43, Evident), and the ratio of viable cells (cell viability) for each sample was calculated. Cell viability was calculated using the following formula. Cell viability (%) = {Number of living cells / (Total number of living and dead cells)} × 100

[0072] 3-5. Evaluation Results and Discussion Table 1 shows the evaluation results for cryopreservation solutions 1-6. [Table 1]

[0073] As shown in Table 1, compared to cryopreservation solution 6 containing DMSO and cryopreservation solution 5 containing high molecular weight hyaluronic acid, cryopreservation solutions 1-4 can suppress foam formation while maintaining a similar cell viability rate. Furthermore, cryopreservation solutions 1 and 4 are colorless and transparent, and have less color than cryopreservation solutions 6 and 5. In addition, although cryopreservation solutions 2 and 3 have a slight color, they suppress foam formation while achieving a higher cell viability rate.

[0074] Figure 1 is a photograph showing the results of the defoaming test. As shown in Figure 1, a lot of foam was generated with cryopreservation solution 6, and with cryopreservation solution 5, some foam was generated and remained, although not as much as with DMSO. In contrast, it was confirmed that no foam was generated with cryopreservation solutions 1 to 4.

[0075] Figure 2 shows the cells after thawing in each cryopreservation solution, with each cell culture vessel containing 4.5 × 10⁶ cells. 3 cells / cm 2This is a phase-contrast image of seeded cells. The culture medium used was the same as that used in the cell culture process described above. As shown in Figure 2, cryopreservation solutions 1 and 4 showed cell adhesion and proliferation that were equivalent to or better than cryopreservation solution 5. [Industrial applicability]

[0076] The cryopreservation solution and cryopreservation method according to the present invention are useful for the cryopreservation of biological samples such as cells and tissues in various fields such as regenerative medicine, livestock farming, and food.

Claims

1. Aqueous solvent and Hyaluronic acid having a weight-average molecular weight of 400 to 8800, and at least one of its salts, At least one amino acid selected from the group consisting of glycine, alanine, valine, asparagine, isoleucine, glutamine, histidine, proline, hydroxyproline, and taurine, including, A cryopreservation solution for biological samples.

2. In the visible light absorption spectrum, the average absorbance at a wavelength of 450 nm is 0.05 or less. The cryopreservation solution according to claim 1.

3. The total concentration of at least one of the hyaluronic acid and its salt is 5 to 60 w / v%. The cryopreservation solution according to claim 1.

4. The molecular weight distribution of at least one of the hyaluronic acid and its salt is 1 to 4. The cryopreservation solution according to claim 1.

5. Further comprising at least one of hyaluronic acid and its salts having a weight-average molecular weight greater than 8800, The cryopreservation solution according to claim 1.

6. The total concentration of hyaluronic acid and its salts with a weight-average molecular weight of 400 to 8800 is greater than the total concentration of hyaluronic acid and its salts with a weight-average molecular weight greater than 8800. The cryopreservation solution according to claim 5.

7. The aforementioned proline, The cryopreservation solution according to claim 1.

8. The concentration of proline is 1 to 7.5 w / v%, The cryopreservation solution according to claim 7.

9. The aforementioned taurine is included, The cryopreservation solution according to claim 1 or 7.

10. The concentration of the taurine is 0.01 to 5 w / v%, The cryopreservation solution according to claim 9.

11. The concentration of dimethyl sulfoxide is 3 w / v% or less. The cryopreservation solution according to claim 1.

12. The total concentration of polyhydric alcohols selected from the group consisting of ethylene glycol, propylene glycol, and glycerol is 0.1 to 15 v / v%. The cryopreservation solution according to claim 1.

13. The total concentration of at least one of the hyaluronic acid and its salt is 10 to 60 w / v%, The cryopreservation solution according to claim 1.

14. The aforementioned biological sample is a cell. The cryopreservation solution according to claim 1.

15. A step of including a biological sample in the cryopreservation solution described in claim 1, A step of freezing the cryopreservation solution containing the biological sample, including, A method for cryopreserving biological samples.

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