Platelet activators

Abstract

To provide an activator that activates a biological sample.SOLUTION: A platelet activator includes a solvent, a polymer having a viscosity average molecular weight of more than 3000 and 500000 or less, the polymer including a monomer having a hydrophilic group as a repeat unit, or a salt thereof, and a saccharide having a viscosity average molecular weight of 3000 or less, or a salt thereof. There is also provided a method for activating a biological sample during cryopreservation.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a platelet activator capable of activating platelets. The present invention also relates to a method for activating a biological sample such as platelets during cryopreservation. [Background technology]

[0002] With the dramatic advances in regenerative medicine research in recent years, cell therapy and other regenerative medicine approaches are being actively pursued not only in humans but also in veterinary medicine. Bone marrow-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells are harvested from living organisms and then expanded in large quantities for use in the aforementioned regenerative medicine and regenerative medicine research. In this case, it is common for excess cells to be cryopreserved and used as needed. There is also a growing demand for a stable supply of such cells.

[0003] In the cryopreservation mechanism of cells, it is known that when ice crystals grow inside cells during the freezing and / or thawing process, the cell membrane and intracellular structures are damaged, and cellular proteins are denatured, resulting in fatal damage to the cells.

[0004] To prevent such intracellular freezing, methods of cryopreserving cells have been used, such as vitrification, which uses a high concentration of cryoprotectant to prevent the formation of ice crystals, and slow freezing, which involves slowly cooling a physiological solution containing cells and a cryoprotectant.

[0005] Dimethyl sulfoxide, for example, is a widely used cryoprotectant. Examples of cryopreservation solutions optimized for the cryopreservation of stem cells include STEM-CELLBANKER (registered trademark) (Zenoac Resources, Inc.) for slow freezing.

[0006] Patent Document 1 describes a cryopreservation solution for biological samples, which contains, in a solvent, a polymer or a salt thereof, the polymer having a viscosity-average molecular weight of more than 3,000 and not more than 500,000, the polymer containing a monomer having a hydrophilic group as a repeating unit, and a saccharide or a salt thereof having a viscosity-average molecular weight of not more than 3,000.

[0007] Platelets are small, disk-shaped, anucleated cells that circulate in mammalian blood and perform blood clotting functions. When vascular injury occurs, platelets adhere and aggregate at the site of vascular injury. The aggregated platelets are activated and exocytose numerous physiologically active substances, including various growth factors and cytokines, contained in their granules, which help promote blood coagulation and tissue regeneration. Platelet-rich plasma (PRP) therapy, which uses platelet-enriched plasma (PRP), is used for wound healing and tissue regeneration. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2020 / 166711 Summary of the Invention [Problem to be solved by the invention]

[0009] In order to release growth factors, cytokines, etc., platelets must be activated.

[0010] The present invention aims to provide a platelet activator that can activate platelets during cryopreservation and that achieves a high survival rate of platelets after freezing and thawing, and a method for activating biological samples such as platelets by cryopreservation. [Means for solving the problem]

[0011] The present invention will be described below.

[0012] As mentioned above, platelets need to be activated in order to release growth factors, cytokines, and the like.

[0013] Platelet activators in the body are collagen, thrombin, and platelet agonists such as adenosine diphosphate (ADP), serotonin, and thromboxane A2, which are released from platelets themselves. In PRP therapy, platelets are activated by adding calcium chloride, freezing and thawing platelets, adding thrombin, or mechanical stimulation.

[0014] In conventional platelet-rich plasma, platelets are activated without being removed, and the plasma is either used immediately after preparation along with growth factors or frozen for storage. Compositions containing growth factors and cytokines obtained from activated platelets typically lose their biological activity within a few hours at room temperature and cannot be stored for long periods even in the presence of platelets. Furthermore, in the absence of platelets, growth factors and cytokines are easily denatured and degraded during frozen storage.

[0015] A typical PRP treatment protocol requires several follow-up injections over a 3- to 9-month period. However, PRP cannot usually be stored between treatments; it is typically prepared from autologous peripheral blood collected for each treatment by centrifugation, extraction (separation), and activation using a kit.

[0016] Therefore, there is a need for a simpler method to activate platelets and release growth factors and cytokines for regenerative medicine applications.

[0017] Patent Document 1 describes that in cryopreservation using the cryopreservation solution of Patent Document 1, cells can be cryopreserved while maintaining a high viability without using substances such as dimethyl sulfoxide, which have been widely used as cryoprotectants in the past but are known to have cytotoxicity and differentiation-inducing properties.

[0018] The present invention provides a platelet activator that can activate platelets during cryopreservation and that achieves a high survival rate of platelets after freezing and thawing, and a method for activating biological samples such as platelets by cryopreservation.

[0019] The present invention relates to a platelet activator comprising a solvent, a polymer having a viscosity-average molecular weight of more than 3,000 and not more than 500,000, the polymer containing a repeating unit of a monomer having a hydrophilic group, or a salt thereof, and a saccharide having a viscosity-average molecular weight of not more than 3,000, or a salt thereof. The salt of the polymer or saccharide is preferably a metal salt, a halogen salt, or a sulfate salt. The metal salt is preferably a salt of an alkali metal or alkaline earth metal. Examples of the alkali metal or alkaline earth metal include sodium, potassium, and calcium. Examples of the halogen include chlorine and bromine.

[0020] Furthermore, the platelet activator of the present invention activates platelets by cryopreserving a biological sample containing the platelet activator of the present invention and platelets.

[0021] In the present invention, the platelet activator preferably contains a polymer or its salt having a viscosity-average molecular weight of more than 3,000 and not more than 500,000, the polymer containing a monomer having a hydrophilic group as a repeating unit, as a major component, and a saccharide or its salt having a viscosity-average molecular weight of not more than 3,000 as a minor component. In this specification, the term "major component" refers to the component with the highest weight ratio among the components of the platelet activator. Components other than the major component in the platelet activator are minor components.

[0022] In the platelet activator of the present invention, platelets are cryopreserved and activated together with the platelet activator. That is, the platelet activator of the present invention activates platelets during cryopreservation. Platelets in a cryopreserved state are vitrified. It is believed that the polymers in the platelet activator of the present invention, particularly the hydrophilic groups in the polymers, are involved in the vitrification of platelets during freezing, the replacement of water around platelet cells with sugars having a molecular weight of 3,000 or less, and the protection of platelet cells from rupture due to freezing. Therefore, it is desirable that the hydrophilic groups in the polymers used in the present invention, which contain repeating units of monomers having hydrophilic groups, are unmodified, or, if modified, that the hydrophilic groups constitute less than 50% of the total number of hydrophilic groups. This is because the cryoprotective effect of the platelet activator is reduced when the degree of hydrophobicity increases due to modification of the hydrophilic groups. Therefore, it is desirable to exclude hydrophobic polymers, such as carboxypolyamino acids, as the main component.

[0023] The platelet activator of the present invention preferably does not contain dimethyl sulfoxide, a cryoprotectant that can function as a differentiation factor, and also preferably does not contain cytotoxic cryoprotectants such as ethylene glycol, because these are harmful to thawed platelets and to the living body receiving the platelets.

[0024] In the present invention, the viscosity average molecular weight of the polymer or its salt is preferably 400,000 or less, particularly 200,000 or less, because the viscosity of the platelet activator before freezing can be adjusted to a low value, making it easy to handle.

[0025] Platelets are activated by the platelet activator of the present invention. As described below, platelets are activated during frozen storage by cryopreserving them in a solvent together with a polymer or a salt thereof having a viscosity-average molecular weight of more than 3,000 and not more than 500,000, the polymer containing a monomer having a hydrophilic group as a repeating unit, and a saccharide or a salt thereof having a viscosity-average molecular weight of not more than 3,000.

[0026] A preferred platelet activator is one in which the monomer having a hydrophilic group is at least one selected from the group consisting of a hydroxyl group and a carboxylic acid group and salts thereof.

[0027] A preferred platelet activator is one in which the polymer further contains, as a repeating unit, a nitrogen-containing monomer having an optionally substituted amino group or an optionally substituted amide group.

[0028] The platelet activator is preferably one in which the polymer is an alternating copolymer of the monomer having a hydrophilic group and the nitrogen-containing monomer.

[0029] A preferred platelet activator is one in which the monomer having a hydrophilic group is a monomer having a hydroxyl group substituted at an equatorial position.

[0030] A platelet activator containing a polymer having a viscosity-average molecular weight of 5,000 or more or a salt thereof is preferred.

[0031] A preferred platelet activator is one in which the polymer comprises multiple sugar residues.

[0032] Preferred are platelet activators in which the saccharide is a monosaccharide, disaccharide, or oligosaccharide.

[0033] Preferred platelet activators are those in which the sugar is glucose, fructose, galactose, or uronic acid in which the alcohol group of any of these is oxidized, or an amino sugar in which the alcohol group is substituted with an amino group, sucrose, a cleavage product of glycosaminoglycan, a constituent monosaccharide of glycosaminoglycan, or a polymer or combination thereof.

[0034] A platelet activator in which the saccharide is glucose, glucuronic acid or N-acetylglucosamine is preferred.

[0035] The polymer or its salt in the platelet activator is preferably present in an amount ranging from 0.1 w / v % to 50 w / v % of the platelet activator.

[0036] The saccharide or its salt in the platelet activator is preferably present in an amount ranging from 0.1 w / v % to 10 w / v % of the platelet activator.

[0037] The present invention also relates to a method for activating a biological sample during cryopreservation, wherein the activation is carried out by a cryoprotectant in cryopreservation.

[0038] The cryoprotectant used in the cryopreservation method for activating a biological sample is preferably a polymer having a viscosity-average molecular weight of more than 3,000 and not more than 500,000, which contains a monomer having a hydrophilic group as a repeating unit, or a salt thereof, and a saccharide having a viscosity-average molecular weight of not more than 3,000, or a salt thereof.

[0039] In the method for activating a biological sample, the biological sample is preferably platelets.

[0040] Cryopreservation is preferably carried out by cooling a sample containing a biological sample and a cryoprotectant by a slow freezing method at a cooling rate of 10°C / min or less, and then storing the sample at -27°C or less.

[0041] The present invention also relates to use of a polymer or a salt thereof having a viscosity-average molecular weight of more than 3,000 and not more than 500,000, the polymer containing a monomer having a hydrophilic group as a repeating unit, and a saccharide or a salt thereof having a viscosity-average molecular weight of not more than 3,000, for activating platelets.

[0042] It is preferred that the polymer or its salt and the saccharide or its salt are cryoprotectants.

[0043] The "viscosity average molecular weight" of the polymer or saccharide used in the present invention can be determined by the following method and calculation formula.

[0044] Intrinsic viscosity measurement: (1) Dissolve a predetermined amount of NaCl in ion-exchanged water at 30°C to prepare a 0.2 M NaCl solution (standard solution). (2) Prepare a stock solution by dissolving a polymer or sugar sample in a standard solution at 30°C. Measure the viscosity of both the standard solution and the stock solution, and adjust the viscosity of the stock solution relative to the standard solution to be 2.0 to 2.4. (3) Dilute the 30°C stock solution 5 / 4, 5 / 3, and 5 / 2 times with the 30°C standard solution. (4) Measure the viscosity of the standard solution, undiluted solution, and diluted solution at 30°C. Use an E-type viscometer to measure the viscosity. (5) The viscosity of the original solution and diluted solution divided by the viscosity of the standard solution is the relative viscosity (η r ) and derive the reduced viscosity based on the following formula: TIFF0007755927000001.tif2146where η sp : Reduced viscosity of polymer or sugar [mL / g], η r : relative viscosity of polymer or sugar [-], C: concentration of polymer or sugar [g / mL]. (6) Plot the relationship between the concentration of the polymer or sugar and the reduced viscosity of the polymer or sugar, and draw an approximate line. The intercept of the approximate line (where the polymer or sugar concentration is 0) is the limiting viscosity.

[0045] Viscosity average molecular weight: The viscosity average molecular weight is calculated from the intrinsic viscosity. TIFF0007755927000002.tif3559The viscosity average molecular weight M is calculated from the above Mark Hoying-Sakurada equation using the intrinsic viscosity derived from the measurement and the values ​​of K and α published in literature, etc.

[0046] K and α are values ​​that vary depending on the type of polymer. The values ​​of K and α are disclosed in many published documents, such as the "Polymer Materials Handbook" (edited by the Society of Polymer Science, Incorporated Association). The viscosity average molecular weight can be calculated using these published values.

[0047] For example, in the case of hyaluronic acid, K = 3.6 × 10 -4 and α = 0.78. For pullulan and gelatin, K = 9 × 10 from the literature. -4 , α=0.5. For example, in the case of dextran, K=6.3×10 -8 , α = 1.4, K = 5.8 × 10 for chondroitin sulfate -4 , α=0.74 can be used.

[0048] In the case of monosaccharides, disaccharides, and compounds that are considered to be monomolecular, the molecular weight is clearly specified from the structural formula, and therefore in the present invention, the molecular weight specified from the structural formula is treated as a hypothetical viscosity average molecular weight.

[0049] The platelet activator of the present invention comprises a solvent, a polymer or salt thereof having a viscosity-average molecular weight of more than 3,000 and not more than 500,000, and a saccharide or salt thereof having a viscosity-average molecular weight of not more than 3,000. The solvent is preferably an aqueous solvent such as water. In particular, it is preferably an isotonic solution in which the salt concentration, saccharide concentration, etc. are adjusted with sodium ions, potassium ions, calcium ions, etc. so that the osmotic pressure is approximately the same as that of body fluids or cellular fluids. Specific examples of such media include water, saline, saline with a buffering effect 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, Ringer's solution, lactated Ringer's solution, acetated Ringer's solution, and bicarbonate Ringer's solution, as well as basal media for animal cell culture such as D-MEM, E-MEM, αMEM, RPMI-1640 medium, Ham's F-12, Ham's F-10, and M-199, and other commercially available media.

[0050] The solvent may also contain calcium chloride, magnesium chloride, magnesium sulfate, potassium chloride, potassium dihydrogen phosphate, sodium hydrogen carbonate, disodium hydrogen phosphate, glucose, sodium chloride, an amino acid, etc. Proline is preferably selected as the amino acid. [Effects of the Invention]

[0051] The platelet activator of the present invention prevents damage to the platelet membrane during cryopreservation by providing solvent vitrification capabilities through the polymer component with a viscosity-average molecular weight of more than 3000 and not more than 500,000, and by inhibiting ice crystal formation and growth near the platelet membrane and providing cell protection through the low-molecular-weight saccharides with a viscosity-average molecular weight of not more than 3000. Therefore, platelets cryopreserved with the platelet activator of the present invention can maintain their morphology and activation after thawing, which is suitable for advanced cell therapy. Specifically, platelet activation is achieved during cryopreservation by the polymer component with a viscosity-average molecular weight of more than 3000 and not more than 500,000 and the low-molecular-weight saccharides with a viscosity-average molecular weight of not more than 3000. Therefore, platelets cryopreserved with the platelet activator of the present invention and thawed platelets are activated platelets suitable for regenerative medicine.

[0052] Furthermore, since the platelet activator of the present invention does not contain highly cytotoxic chemicals such as DMSO or ethylene glycol used for cryopreservation, cryopreserved products containing activated platelets cryopreserved using the platelet activator can be directly applied to cell therapy after thawing. However, cryopreserved products can contain low concentrations of chemicals that do not impair the function of activated platelets or their efficacy as cell therapy agents.

[0053] Furthermore, depending on the site of administration, cryopreserved materials may promote repair or regeneration effects.

[0054] Furthermore, because the platelet activator of the present invention does not contain serum and / or serum-derived proteins, platelets frozen using the platelet activator of the present invention will not be contaminated with bacteria or viruses. However, it is possible to add proteins that are not contaminated with bacteria or viruses.

[0055] Furthermore, in the present invention, platelets are activated with a cryoprotectant simultaneously with cryopreservation, eliminating the need for a platelet activator to activate the platelets after freezing and thawing. This reduces the likelihood that biologically active proteins, such as growth factors, will be degraded by the platelet activator. For example, bovine proteins such as thrombin, which are used for platelet activation, may be pathogens or cause allergic reactions, but such substances are unnecessary for the platelet activation of the present invention.

[0056] Furthermore, in the present invention, cryopreservation is performed by cooling a liquid sample containing a biological sample and a cryoprotectant. However, this cooling does not require the high cooling rate required for cell protection, which is typically required to vitrify the solvent in the liquid sample. Preservation of frozen cryopreserved samples also does not require low temperatures, such as those achieved by using liquid nitrogen. Therefore, the platelet activator of the present invention can efficiently and cost-effectively cryopreservate biological samples. By using the platelet activator of the present invention, activated biological samples can be provided efficiently, at the timing and in the amount required. For example, in PRP therapy, the number of blood draws required for each treatment can be reduced. Frozen platelets can be thawed and used at the time of treatment. Furthermore, cryopreservation using the platelet activator of the present invention activates the platelets in the cryopreserved samples, thereby significantly improving the therapeutic effect.

[0057] Alternatively, such platelets and released intracellular vesicles may be cryopreserved / activated and provided to a medical institution. In this case, the platelets and released intracellular vesicles may be provided in a frozen state or in a freeze-dried state, which is obtained by sublimating the solvent from the frozen state and powdering them. [Brief explanation of the drawings]

[0058] [Figure 1] FIG. 1 shows the activation of platelets that have been cryopreserved and then frozen and thawed using the platelet activator of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0059] The platelet activator of the present invention comprises a solvent, a polymer or a salt thereof having a viscosity-average molecular weight of more than 3,000 and not more than 500,000, and a saccharide or a salt thereof having a viscosity-average molecular weight of not more than 3,000. The polymer of the present invention comprises a monomer having a hydrophilic group as a repeating unit.

[0060] The viscosity average molecular weight of the polymer or salt thereof of the present invention is desirably 400,000 or less, particularly 200,000 or less, because the viscosity of the platelet activator of the present invention before freezing can be adjusted to a low value, making it easy to handle.

[0061] The "viscosity average molecular weight" of the polymer or saccharide of the present invention means a value calculated by the following method and formula.

[0062] The method for measuring the intrinsic viscosity and the method for calculating the viscosity average molecular weight using the intrinsic viscosity are described below. Intrinsic viscosity measurement: (1) Dissolve a predetermined amount of NaCl in ion-exchanged water at 30°C to prepare a 0.2 M NaCl solution (standard solution). (2) Prepare a stock solution by dissolving the polymer or sugar sample in a standard solution at 30°C. If the polymer or sugar sample is obtained in solution, remove the solvent from the solution to obtain the solid portion. For mixed samples of polymers and sugars, or mixed samples containing multiple polymers or multiple sugars, separate and fractionate each substance, then remove the solvent from each substance to obtain the polymer or sugar sample. If the polymer and / or sugar are unknown, identify them using HPLC, LC-MS, LC-IR, or other methods. If multiple unknown polymers and / or sugars are included, separate and fractionate each component, identify the substance for each polymer and / or sugar using HPLC, LC-MS, LC-IR, or other methods, and calculate the viscosity-average molecular weight as described below. Even if the polymer or sugar contains impurities that do not affect the viscosity (e.g., metal salts), the mixture can be considered a polymer or sugar sample. If the solution contains impurities that affect the calculation of the viscosity average molecular weight, remove the impurities or separate the polymers and sugars before measuring. Measure the viscosity of the standard solution and the stock solution, and adjust the relative viscosity of the stock solution to 2.0 to 2.4 compared to the standard solution. (3) Dilute the 30°C stock solution 5 / 4, 5 / 3, and 5 / 2 times with the 30°C standard solution. (4) Measure the viscosity of the standard solution, undiluted solution, and diluted solution at 30°C. Use an E-type viscometer to measure the viscosity. (5) The viscosity of the original solution and diluted solution divided by the viscosity of the standard solution is the relative viscosity (η r ) and derive the reduced viscosity based on the following formula: TIFF0007755927000003.tif2146where η sp : Reduced viscosity of polymer or sugar [mL / g], η r : relative viscosity of polymer or sugar [-], C: concentration of polymer or sugar [g / mL]. (6) Plot the relationship between the concentration of the polymer or sugar and the reduced viscosity of the polymer or sugar, and draw an approximate line. The intercept of the approximate line (where the polymer or sugar concentration is 0) is the limiting viscosity. Viscosity average molecular weight: The viscosity average molecular weight is calculated from the intrinsic viscosity. TIFF0007755927000004.tif3559The viscosity average molecular weight M can be calculated from the above Mark Hoeing-Sakurada equation using the intrinsic viscosity determined by measurement and the K and α values ​​published in literature. In the case of hyaluronic acid, K = 3.6 x 10 -4 and α = 0.78 to calculate the viscosity average molecular weight M. For the pullulan and gelatin used in the examples, K = 9 × 10 -4 , α=0.5, and for dextran, K=6.3×10 -8 , α = 1.4, and for chondroitin sulfate, K = 5.8 × 10 -4 , α = 0.74, for carboxypolylysine, K = 2.78 × 10 -5 , α=0.87 is used.

[0063] K and α are values ​​that vary depending on the type of polymer. The values ​​of K and α are disclosed in many published documents, such as the "Polymer Materials Handbook" (edited by the Society of Polymer Science, Incorporated Association). The viscosity average molecular weight is calculated using these published values.

[0064] In the present invention, the molecular weight calculated by this method is referred to as the viscosity average molecular weight. In the case of monosaccharides, disaccharides, and compounds that are considered to be monomolecular molecules, such as sucrose and glucuronic acid, the molecular weight is clearly specified from the structural formula, and therefore the molecular weight specified from the structural formula is treated as the viscosity average molecular weight.

[0065] The polymers contained in the platelet activator of the present invention, which have a specific molecular weight and numerous hydrophilic groups, trap water molecules of the solvent within a matrix formed by the polymer chains during the cooling process for cryopreservation. Because the polymer chains contain hydrophilic groups, the molecular motion of the water solvent is restricted during cooling, allowing the water to solidify and / or freeze in a vitrified state without crystallizing. That is, in a cryopreserved product containing platelets that has been cryopreserved using the platelet activator of the present invention, the intracellular contents of the platelets are dehydrated and vitrified by the action of the polymer chains of the present invention. Therefore, the preparation of a cryopreserved product using the platelet activator of the present invention does not require the increase in the concentration of solutes (cryoprotectants) or the increase in the cooling rate, as in conventional vitrification methods. In the cryopreserved product containing platelets obtained using the platelet activator of the present invention, the action of the polymer chains suppresses the formation of ice crystals within the platelets. The difference in osmotic pressure between the inside and outside of the cells, which is required in conventional vitrification methods for intracellular dehydration, is not required during the preparation of the cryopreserved product, thereby reducing the osmotic shock in cells during freezing, a problem associated with conventional vitrification methods. Furthermore, since recrystallization does not occur when the frozen storage product is thawed, it is believed that the platelets contained in the frozen storage product will be less damaged by thawing.

[0066] The viscosity-average molecular weight of the polymer of the present invention is greater than 3,000 and not greater than 500,000. This level of viscosity-average molecular weight stabilizes the amorphous, vitreous state in the frozen state of a cryopreserved product obtained using the platelet activator of the present invention. Platelet cells contained in the cryopreserved product can remain stable. Therefore, the survival rate of platelet cells after thawing the cryopreserved product is high. When the viscosity-average molecular weight of the polymer is 3,000 or less, vitrification may not occur well. Furthermore, when the viscosity-average molecular weight of the polymer is greater than 500,000, the viscosity significantly increases, and the solubility in solvents decreases. Problems such as foaming of the solution before cryopreservation and poor handling may occur. The viscosity-average molecular weight is preferably 5,000 or more. Furthermore, polymers with a viscosity-average molecular weight of 400,000 or less, or even 200,000 or less, are preferred, with 150,000 or less being particularly preferred. This is because the viscosity of the platelet activator can be adjusted low, making it easier to handle.

[0067] The polymer of the present invention is a polymer containing a repeating unit of a monomer having a hydrophilic group. Examples of the hydrophilic group include a hydroxyl group and a carboxylic acid group and its salt. The polymer of the present invention 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 polymer of the present invention preferably has a hydroxyl group at an equatorial position within its structure. This is believed to enable the water solvent to be more effectively trapped within the matrix formed by the polymer chains during freezing.

[0068] An example of a monomer having a hydrophilic group is a sugar residue. In this case, the polymer of the present invention may be a polymer containing repeating units of sugar residues linked by glycosidic bonds, or derivatives thereof. Examples of sugar residues include, but are not limited to, monosaccharides, or monosaccharides in which the hydroxyl and / or hydroxymethyl groups of monosaccharides have been substituted, such as monosaccharides in which the hydroxyl and / or hydroxymethyl groups have been substituted with at least one substituent selected from the group consisting of a carboxyl group, an amino group, an N-acetylamino group, a sulfoxy group, a methoxycarbonyl group, and a carboxymethyl group.

[0069] Monosaccharides include triose, tetrose, pentose, hexose, and heptose. For example, pentoses include ribose, arabinose, xylose, lyxose, xylulose, ribulose, and deoxyribose. Hexoses include glucose, mannose, galactose, fructose, sorbose, tagatose, fucose, fuculose, and rhamnose.

[0070] For example, monosaccharides substituted with a carboxyl group include uronic acid. Examples of uronic acids include glucuronic acid, iduronic acid, mannuronic acid, and galacturonic acid. Examples of monosaccharides substituted with an amino group include amino sugars. Examples of amino sugars include glucosamine, galactosamine, mannosamine, and muramic acid. Examples of monosaccharides substituted with an N-acetylamino group include N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, and N-acetylmuramic acid. Examples of monosaccharides substituted with a sulfooxy group include galactose-3-sulfate. Examples of monosaccharides with multiple substituents include N-acetylglucosamine-4-sulfate, iduronic acid-2-sulfate, glucuronic acid-2-sulfate, N-acetylgalactosamine-4-sulfate, neuraminic acid, and N-acetylneuraminic acid.

[0071] For example, the polymer of the present invention may be a polymer containing the above-mentioned monosaccharides as repeating units. For example, the polymer of the present invention may be a polymer containing an optionally substituted pentose, hexose, or uronic acid, or a combination thereof, as repeating units. The polymer of the present invention may also be an alternating copolymer of a monomer having a hydrophilic group and a nitrogen-containing monomer. The nitrogen-containing monomer may be, for example, an amino sugar. In this case, for example, the polymer of the present invention may be a glycosaminoglycan. Alternatively, the polymer may be a sulfated polysaccharide in which one or more hydroxyl groups are substituted with sulfoxy groups. Examples of the polymer of the present invention include, but are not limited to, hyaluronic acid, dextran, pullulan, and chondroitin sulfate.

[0072] The polymers used in the present invention may be naturally occurring, chemically synthesized, or commercially available. Naturally occurring or commercially available polymers with larger molecular weights may be subjected to hydrolysis, enzymatic treatment, subcritical treatment, or other treatments to obtain cleavage products, and the molecular weights may then be adjusted to produce the polymers of the present invention. Furthermore, each monomer may be naturally occurring, a naturally occurring monomer that has been modified or substituted, or a chemically synthesized monomer. For example, preferably, the monomers contained in the polymers of the present invention are biological components. These monomers are advantageous because they have low cytotoxicity and can be directly cryopreserved and used in cell preparations.

[0073] The hydrophilic groups of the polymer of the present invention are desirably unmodified, or if modified, their number is 50% or less of the total number of hydrophilic groups, i.e., no substituents are introduced into the polymer chain, or if introduced, their number is 50% or less of the total number of hydrophilic groups. It is believed that the hydrophilic groups of the polymer, particularly OH, NH, and COOH groups, contribute to the protection of cells frozen using the platelet activator of the present invention, the vitrification of the solvent, and the replacement of sugar with water around the platelet cells. Therefore, it is believed that not modifying these functional groups is advantageous for improving the survival rate of platelet cells after thawing.

[0074] Furthermore, the hydrophilic groups of the polymers of the present invention are thought to hold low-molecular-weight sugars by hydrogen bonds, and the presence of such polymers holding low-molecular-weight sugars around platelet cells is presumed to promote the replacement of water molecules and sugars near the cell membrane of platelet cells. Therefore, if the hydrophilic groups are modified, the hydrophilic groups' ability to hold low-molecular-weight sugars is reduced, and even if low-molecular-weight sugars coexist, they may not sufficiently contribute to improving the survival rate of platelets. Therefore, modifying OH groups or NH groups with carboxylic acids or the like may not be desirable.

[0075] The platelet activator of the present invention contains a saccharide or its salt having a viscosity-average molecular weight of 3,000 or less. This saccharide displaces water molecules near the cell membrane of platelet cells, thereby inhibiting the formation and growth of ice crystals near the cell membrane. As a result, cell membrane damage in platelets frozen during cryopreservation using the platelet activator of the present invention can be significantly suppressed. That is, the saccharide or its salt used in the present invention can function as a component for protecting platelet cells. The saccharide of the present invention can be, for example, a monosaccharide, disaccharide, or oligosaccharide having a molecular weight of 3,000 or less, preferably 2,000 or less, and more preferably 1,000 or less.

[0076] Such sugars include, for example, the monosaccharides described above as monomers constituting the polymers of the present invention. For example, sugars include glucose, fructose, galactose, or uronic acids in which the alcohol group of these sugars is oxidized, or amino sugars in which the alcohol group is substituted with an amino group, sucrose, trehalose, or polymers or combinations thereof. Furthermore, sugars may be, for example, fragments of the polymers used in the present invention, such as hyaluronic acid, dextran, pullulan, or chondroitin sulfate. While not particularly limited as long as the effects of the present invention are not impaired, sugars may be, for example, cleavage products (fragments) of glycosaminoglycans, i.e., monosaccharides, disaccharides, or oligosaccharides constituting glycosaminoglycans.

[0077] Preferably, saccharide is glucose or hyaluronic acid cleavage product.Therefore, preferably, saccharide of the present invention is glucose, glucuronic acid or N-acetylglucosamine, or disaccharide or oligosaccharide thereof.Preferably, saccharide can be glucuronic acid or its modified compound, or its disaccharide or oligosaccharide.

[0078] The term "cleavage product" as used herein refers to a compound having a smaller molecular weight than the original polymer, which is thought to be obtained when a polymer is subjected to treatment such as hydrolysis, enzymatic treatment, or subcritical treatment. That is, the polymer of the present invention may be a polymer having a viscosity-average molecular weight of more than 3,000 and not more than 500,000, obtained by treating a larger polymeric compound, as described above, and the saccharide of the present invention may be a saccharide having a viscosity-average molecular weight of not more than 3,000, obtained by treating the polymer of the present invention. The cleavage product may be a monomer that is a component of the original polymer, and / or polymers of the monomers with various degrees of polymerization, and / or a mixture thereof.

[0079] "Subcritical processing" refers to contacting a raw material to be extracted with a subcritical fluid (extraction solvent) that has been brought to a subcritical state under predetermined temperature and pressure conditions. For example, water is neither liquid nor gaseous when the pressure is raised to 22.12 MPa or higher and the temperature to 374.15°C or higher. This point is called the critical point of water, and hot water at temperatures and pressures near the critical point is called subcritical water. The hydrolysis action of this subcritical water can be used to obtain desired components from the raw material to be extracted. Conditions for subcritical processing in the present invention include, for example, a temperature of 150°C or higher and 350°C or lower, and the subcritical processing pressure can be set to a value equal to or higher than the saturated vapor pressure at each temperature, e.g., 0.5 MPa or higher and 25 MPa or lower. After subcritical processing, components with molecular weights below a predetermined value are separated and recovered and can be used as the cleavage product in the present invention. Furthermore, there are no particular limitations on the hydrolysis or enzymatic processing, and commonly used reagents and processing methods can be used without any problems.

[0080] The polymer and saccharide of the present invention may be simultaneously obtained by a single subcritical treatment. That is, the polymer and saccharide of the present invention may be subcritically treated products of a polymer compound having a first molecular weight distribution in a molecular weight range of more than 3,000 and not more than 500,000, as measured by viscosity average molecular weight, and a second molecular weight distribution in a molecular weight range of not more than 3,000, as measured by viscosity average molecular weight.

[0081] Salts of the polymer or saccharide of the present invention include metal salts, halogen salts, and sulfate salts. Metal salts are preferably salts of alkali metals or alkaline earth metals. Examples of alkali metals or alkaline earth metals include sodium, potassium, and calcium. Examples of halogens that can be used include chlorine and bromine.

[0082] The platelet activator of the present invention includes a polymer or its salt having a viscosity-average molecular weight greater than 3,000 and not greater than 500,000, the polymer containing a repeating unit of a monomer having a hydrophilic group, and a saccharide or its salt having a viscosity-average molecular weight not greater than 3,000, which are cryoprotectants for cryopreservation. Specifically, the platelet activator of the present invention contains a cryoprotectant that combines a polymer that can suppress rupture of biological samples such as cells due to ice crystal formation by trapping water molecules with polymer chains during freezing, preventing ice crystal formation in the solvent and vitrifying the sample, and a low-molecular-weight saccharide that replaces water molecules near the boundary tissue of platelet cells with the solvent, thereby inhibiting ice crystal formation and growth near the cell membrane of platelet cells and protecting the cell membrane of platelet cells. The cryoprotectant of the present invention effectively suppresses ice crystal formation during freezing and recrystallization during thawing, thereby providing the resulting cryopreserved product with a high cryopreservation effect not obtainable with conventional techniques. At the same time, the cryoprotectant of the present invention can activate platelets contained in the cryopreserved product during cryopreservation. Furthermore, even after thawing of the cryopreserved material, the activated state of the platelets is maintained, and the platelets retain the biological activity and therapeutic effect of activated platelets.

[0083] In the platelet activator of the present invention, the polymer or its salt is present in the platelet activator in an amount ranging from 0.1 w / v% to 50 w / v% of the platelet activator. An amount less than 0.1 w / v% may result in insufficient vitrification of the solvent portion of the frozen cryopreserved product. An amount greater than 50 w / v% may result in excessively high viscosity of the solution containing the polymer or its salt, potentially resulting in poor handling. For example, the polymer or its salt is preferably present in an amount of 5 w / v% or more, with 10 w / v% or more being particularly preferred. The concentration of the polymer or its salt is preferably 20 w / v% or less. The amount of the polymer or its salt contained in the platelet activator of the present invention may be 5 w / v% or more and 20 w / v% or less.

[0084] In the platelet activator of the present invention, the saccharide or its salt is present in the platelet activator at a concentration ranging from 0.1 w / v% to 10 w / v% of the platelet activator. If the saccharide or its salt is present in the platelet activator at less than 0.1 w / v%, the effects of the present invention may not be fully achieved. Furthermore, even if the saccharide or its salt is present in the platelet activator at 10 w / v% or more of the platelet activator, it is difficult to achieve additional effects as a cell-protecting component. The weight ratio of polymer to saccharide in the platelet activator of the present invention is preferably 1:1 to 500:1, more preferably 1:1 to 50:1, and optimally 1:1 to 20:1.

[0085] In the cryopreservation of platelets using an activator according to the present invention, a liquid sample containing a biological sample, a solvent, and a cryoprotectant (a polymer having a viscosity-average molecular weight of more than 3,000 and not more than 500,000, the polymer containing a monomer having a hydrophilic group as a repeating unit, or a salt thereof, and a saccharide having a viscosity-average molecular weight of not more than 3,000, or a salt thereof) is cryopreserved. The biological sample of the present invention is preferably platelets. Platelets are separated from collected blood or pre-chilled blood. There are no limitations on the means for separating platelets from blood (whole blood), and any known means can be used. For example, blood is centrifuged to separate it into a fraction containing blood cells and platelet-rich plasma (PRP). The platelet-rich plasma (PRP) is then centrifuged to separate it into platelets and platelet-poor plasma (PPP).

[0086] The solvent contained in the platelet activator of the present invention may be, for example, an isotonic solution in which the salt concentration or sugar concentration is adjusted with sodium ions, potassium ions, calcium ions, etc. so that the osmotic pressure is approximately the same as that of body fluids or cell fluids. Specific examples include, but are not limited to, water, physiological saline, physiological saline with a buffering effect such as phosphate buffered saline (PBS), Dulbecco's phosphate buffered saline, Tris buffered saline (TBS), HEPES-buffered saline, balanced salt solutions such as Hank's balanced salt solution (HBSS), Ringer's solution, lactate Ringer's solution, acetate Ringer's solution, and bicarbonate Ringer's solution. Furthermore, the solvent may contain other optional components, such as isotonic agents, chelating agents, solubilizers, pH adjusters, and additives commonly used in cell culture media, as long as they do not impair the effects of the present invention.

[0087] The platelet activator of the present invention may further contain, as an optional component, a cryoprotective support substance that enhances the cryoprotective effect of the cryoprotectant. The cryoprotective support substance is a substance other than saccharides or their salts having a viscosity-average molecular weight of 3000 or less. Examples of such substances include amino acids known to form ice nuclei in solution at temperatures higher than the freezing point of intracellular water. Examples of such amino acids include glycine, alanine, valine, asparagine, isoleucine, glutamine, proline, and histidine. Furthermore, the cryoprotective support substance may be 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. Such a cryoprotective support substance may be contained in the platelet activator of the present invention at a concentration of, for example, 0.1 w / v% or more and 10 w / v% or less.

[0088] In this specification, the term "optional component" refers to a component that may or may not be included.

[0089] For example, the solvent for the platelet activator of the present invention may be a 5% aqueous glucose solution, etc. Alternatively, the 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, or M-199.

[0090] Cryopreservation using the platelet activator of the present invention results in minimal cellular damage to frozen platelets. This is because a biological sample can be effectively frozen to give a frozen product by cooling it together with the platelet activator of the present invention to −27°C or below, for example, at a cooling rate of approximately 10°C / min or less. Therefore, obtaining a frozen product using the platelet activator of the present invention does not require a high cooling rate, which is required for known vitrification methods to reduce osmotic shock during cryopreservation. For example, the biological sample and the platelet activator of the present invention can be mixed, transferred to a freezing container, or the like, and placed in a deep freezer at −80°C. This allows the biological sample to be cryopreserved while maintaining a high viability. No special procedures or equipment are required to obtain a frozen product. A frozen product containing activated platelets can be easily obtained. The cryopreservation temperature range is not limited to −27°C or below, but the upper limit is preferably −70°C or below, and preferably −80°C or below. The lower limit is preferably −196°C or above, and preferably −150°C or above.

[0091] The storage period of the frozen preserved material containing a biological sample of the present invention in a frozen state is not particularly limited, and may be, for example, 1 week or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 1 year or more, or more. Even after such long-term storage in a frozen state, platelets are activated and maintain a high survival rate after thawing.

[0092] The polymer of the present invention, which has a viscosity-average molecular weight greater than 3,000 and less than 500,000 and contains a monomer having a hydrophilic group as a repeating unit, is a non-permeating cryoprotective reagent during cryopreservation, and is therefore thought to have low cytotoxicity to cryopreserved cells. Furthermore, in the present invention, the sugars used together with the polymer function to protect cell membranes during cryopreservation. The same polymer and sugars stimulate and activate platelets, resulting in cryopreservation of activated platelets. Therefore, the present invention provides a method for activating a biological sample during cryopreservation, characterized in that activation is performed using a cryoprotectant during cryopreservation. In this method, cryopreservation can be performed using a slow freezing method, making it possible to easily prepare activated biological samples.

[0093] As the polymer of the present invention having a viscosity-average molecular weight of more than 3,000 and not more than 500,000, which contains a monomer having a hydrophilic group as a repeating unit, a polymer that is a biological component is more preferred. By using such a polymer or a salt thereof, activated platelets can be directly applied to cell therapy after thawing a cryopreserved sample. For example, a preferred polymer of the present invention is hyaluronic acid. In particular, it is hyaluronic acid having a viscosity-average molecular weight of not more than 400,000, preferably not more than 200,000. More particularly, it includes hyaluronic acid having a viscosity-average molecular weight of more than 3,000, more preferably more than 5,000, and not more than 60,000, more preferably not more than 20,000. [Example]

[0094] The present invention will be specifically described based on examples, but the present invention is not limited to these.

[0095] <Preparation of cryoprotectant> In a 2L pressure vessel, high molecular weight hyaluronic acid (manufactured by Shanghai Easier Industrial Development Co., Ltd.) with an average molecular weight of 1 million was mixed with water at a ratio of 20:100, and the mixture was subjected to subcritical treatment at a temperature of 175°C, a pressure of 0.89 MPa, and a treatment time of 3 minutes. The subcritical treatment product was then freeze-dried or spray-dried. This resulted in a mixture of high molecular weight hyaluronic acid with an intrinsic viscosity of 0.49 dL / g and a viscosity-average molecular weight of 10,000, and low molecular weight hyaluronic acid with an intrinsic viscosity of 0.08 dL / g and a viscosity-average molecular weight of 1,000, i.e., the high molecular weight and low molecular weight sugars used as cryoprotectants in the present invention.

[0096] <Preparation of platelets> Platelets were isolated from fresh blood collected from C57BL / 6 mice (15 weeks old, male). Specifically, blood was collected using a syringe containing acid-citrate-dextrose formula A and transferred to a 2 mL tube containing buffered saline glucose citrate (pH 7.3). The collected blood was centrifuged at 1500 rpm at 20°C for 5 minutes to separate blood cells and platelet-rich plasma (PRP). The PRP was then collected and centrifuged at 1000 rpm at 20°C for 5 minutes to remove as many blood cells as possible. The PRP was then centrifuged at 3600 rpm at 20°C for 5 minutes to separate platelets and platelet-poor plasma (PPP), and only the platelets were collected.

[0097] <Platelet sample preparation> The separated and collected platelets were sedimented by centrifugation and then diluted at 2 × 10 in Tyrode buffer (composition: NaCl 8 g / L, KCl 0.2 g / L, CaCl2 0.2 g / L, MgCl2·6H2O 0.1 g / L, NaH2PO4·H2O 0.05 g / L, glucose 1 g / L, NaHCO3 1 g / L). 8 The platelets were resuspended at a concentration of 1000 / mL to prepare a platelet solution.

[0098] <Activation of fresh platelets and measurement of activation> Comparison Example 1 The platelet solution obtained above was diluted to 1 × 10 7 Platelets were collected so that each platelet count was 1000 and diluted with an equal volume of Tyrode's buffer. As a negative control, 20 μL of Tyrode's buffer was added.

[0099] After replacing the solvent with Tyrode's buffer containing 1 mM CaCl2, the cell surface markers CD41 / 61 and CD62-P, which serve as platelet markers and activation indicators, respectively, were evaluated by flow cytometry (BD FACSCanto II). Fluorescent dye PE-conjugated antibodies (anti-CD41 / 61) and FITC-conjugated antibodies (anti-P-selectin / CD62-P) (EMFRET Analytics GmbH & Co. KG, catalog number: D200) were used.

[0100] Comparative Example 2 As in Comparative Example 1, the platelet solution was diluted to 1 × 10 7 Platelets were collected so that each platelet count was 1000 and diluted with an equal volume of Tyrode's buffer. The platelets were activated by adding 20 μL of 1 U / mL thrombin (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0101] As in Comparative Example 1, CD41 / 61 and CD62-P were evaluated.

[0102] <Measurement of frozen platelet activation> Example 1 One hundred grams of the cryoprotectant, a mixture of high molecular weight and low molecular weight saccharides, prepared as described above, was added to one liter of water for injection containing 0.14 g / L calcium chloride, 0.10 g / L magnesium chloride hexahydrate, 0.10 g / L magnesium sulfate heptahydrate, 0.40 g / L potassium chloride, 0.06 g / L potassium dihydrogen phosphate, 0.35 g / L sodium bicarbonate, 0.048 g / L disodium hydrogen phosphate, 11 g / L D(+)-glucose, and 9 g / L sodium chloride, and 10 g / L proline to obtain a platelet activator (containing 10% by weight of a mixture of high molecular weight and low molecular weight saccharides, with a viscosity-average molecular weight of 10,000 and a viscosity-average molecular weight of 1,000, and a low molecular weight hyaluronic acid ratio of 10:1).

[0103] Platelet solution 1 x 10 7 Platelets were collected so that each sample was 1000 pieces, and diluted with an equal volume of the platelet activator prepared above. The obtained sample was placed in a cryopreservation container (Mr. Frosty), frozen in a deep freezer at a cooling rate of 1°C / min, and stored frozen until use.

[0104] After about one month of cryopreservation, the frozen platelets were quickly thawed in a water bath at 37°C, and CD41 / 61 and CD62p were evaluated in the same manner as in the measurement of platelet activation in Comparative Example 1.

[0105] Comparative Example 3 As in Comparative Example 1, the platelet solution was diluted to 1 × 10 7 Platelets were collected so that each sample amounted to 1000 pieces, and diluted with an equal volume of Tyrode's buffer. The obtained sample was placed in a cryopreservation container (Mr. Frosty), frozen in a deep freezer at a cooling rate of 1°C / min, and stored frozen until use.

[0106] After about one month of cryopreservation, the frozen platelets were quickly thawed in a water bath at 37°C, and CD41 / 61 and CD62p were evaluated in the same manner as in the measurement of platelet activation in Comparative Example 1.

[0107] Comparative Example 4 Platelet solution 1 x 107 Platelets were collected and diluted with an equal volume of a commercially available DMSO-containing cryopreservation solution (STEM-CELLBANKER® GMP grade (containing DMSO), manufactured by Zenoac Resources, Inc.; a preservation solution presumably prepared by mixing 100 mL of DMSO with 5 g of sodium carboxymethylcellulose (molecular weight 760,000) dissolved in 750 mL of distilled water and 30.0 g of glucose, 0.8 g of sodium bicarbonate, 0.36 g of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and 1.576 g of phosphate buffer solution dissolved in 150 mL of distilled water). The resulting samples were placed in a cryopreservation container (Mr. Frosty) and frozen at a cooling rate of 1°C / min in a deep freezer. The samples were then stored frozen until use.

[0108] After about one month of cryopreservation, the frozen platelets were quickly thawed in a water bath at 37°C, and CD41 / 61 and CD62p were evaluated in the same manner as in the measurement of platelet activation in Comparative Example 1.

[0109] The detection results by flow cytometry in Comparative Examples 1 to 4 and Example 1 are shown in FIG.

[0110] In the results shown in FIG. 1 , Comparative Examples 1 to 4 and Example 1 respectively show unfrozen, unactivated platelets (Comparative Example 1), unfrozen thrombin-activated platelets (Comparative Example 2), platelets cryopreserved in the absence of a cryoprotectant and then thawed (Comparative Example 3), platelets cryopreserved with the platelet activator of the present invention, i.e., in the presence of the cryoprotectant of the present invention, and then thawed (Example 1), and platelets cryopreserved using a commercially available cryopreservation solution containing DMSO and then thawed (Comparative Example 4).

[0111] As shown in Figure 1, platelets cryopreserved with the platelet activator of the present invention showed enhanced cell surface translocation (expression) of CD62-P and CD41 / 61. High platelet activation was confirmed by cryopreservation using the platelet activator of the present invention. This level of activation was higher than the results of thrombin activation of unfrozen platelets (Comparative Example 2) and cryopreservation under conditions not containing the cryoprotectant of the present invention (Comparative Example 3). Such activation was not observed when cryopreservation was performed using a commercially available cryopreservation solution.

[0112] The above results demonstrate that the platelet activator of the present invention has the remarkable effect of enabling stable cryopreservation of biological samples with high cell viability while achieving high activation. The platelet activator of the present invention does not contain cytotoxic cryoprotectants such as DMSO or ethylene glycol, and / or serum or serum-derived proteins. Furthermore, cryopreservation with the platelet activator of the present invention simultaneously preserves and activates biological samples. Therefore, from the viewpoints of biological activity and ease of use, the platelet activator of the present invention may be advantageously applied to regenerative medicine.

Claims

1. a solvent; Hyaluronic acid or a salt thereof having a viscosity average molecular weight of more than 3,000 and not more than 500,000; Hyaluronic acid or its salt having a viscosity average molecular weight of 3000 or less A platelet activator comprising: A platelet activator that activates platelets during cryopreservation.

2. The platelet activator according to claim 1, wherein the hyaluronic acid or its salt having a viscosity average molecular weight of more than 3,000 and not more than 500,000 is present in the platelet activator in an amount ranging from 0.1 w / v% to 50 w / v% of the platelet activator.

3. The platelet activator according to claim 1 or 2, wherein the hyaluronic acid or its salt having a viscosity average molecular weight of 3000 or less is present in the platelet activator in an amount ranging from 0.1 w / v% to 10 w / v% of the platelet activator.

4. 1. A method for activating platelets during cryopreservation, comprising: The cryoprotectant for the cryopreservation is hyaluronic acid or a salt thereof having a viscosity average molecular weight of more than 3000 and not more than 500,000, and hyaluronic acid or a salt thereof having a viscosity average molecular weight of not more than 3000, The method wherein the activation is performed by the cryoprotectant during the cryopreservation.

5. 5. The method according to claim 4, wherein the cryopreservation is carried out by cooling the sample containing the platelets and the cryoprotectant by a slow freezing method at a cooling rate of 10°C / min or less, and then storing the sample at -27°C or less.

6. To activate platelets during cryopreservation, Use of hyaluronic acid or its salt having a viscosity average molecular weight of more than 3,000 and not more than 500,000, and hyaluronic acid or its salt having a viscosity average molecular weight of not more than 3,000 as a cryoprotectant.

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