JP7755927B2 - Platelet activators - Google Patents

Description

The present invention relates to a platelet activator capable of activating platelets. The present invention also relates to a method for activating biological samples such as platelets during cryopreservation.

With the dramatic advances in regenerative medicine research in recent years, cell therapy and other regenerative medicine techniques 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 collected from living organisms and then grown in large quantities for use in the regenerative medicine and regenerative medicine research described above. In this case, it is common for surplus cells to be cryopreserved and used as needed. Demand for a stable supply of such cells is also increasing.

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

To prevent this intracellular freezing, methods of cryopreserving cells have included 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.

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.), which is designed for slow freezing.

Patent Document 1 describes a cryopreservation solution for biological samples, which contains, in 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, 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.

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 contained in their granules, including various growth factors and cytokines, which help promote blood clotting and tissue regeneration. Platelet-rich plasma (PRP) therapy, which uses platelet-enriched plasma (PRP), is used for wound healing and tissue regeneration.

International Publication No. 2020/166711

Platelets need to be activated in order to release growth factors, cytokines, etc.

The present invention aims to provide a platelet activator that can activate platelets during cryopreservation and that has a high platelet survival rate after freezing and thawing, as well as a method for activating biological samples such as platelets through cryopreservation.

The present invention is explained below.

As mentioned earlier, platelets need to be activated in order to release growth factors, cytokines, etc.

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.

In conventional platelet-rich plasma, platelets are activated without being removed, and are either used immediately after preparation along with growth factors, or stored frozen for later use. 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.

A typical PRP therapy protocol requires several follow-up injections over a period of three to nine months. However, PRP cannot usually be stored between treatments; instead, autologous peripheral blood is drawn for each treatment and typically prepared using a kit that centrifuges, extracts (separates), and activates the blood.

Therefore, a simpler method is needed to activate platelets and release growth factors and cytokines for use in regenerative medicine.

Patent Document 1 describes that when cells are cryopreserved using the cryopreservation solution described in Patent Document 1, they can be cryopreserved while maintaining a high viability without using substances such as dimethyl sulfoxide, which have traditionally been widely used as cryoprotectants but are known to be cytotoxic and induce differentiation.

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

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.

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.

In the present invention, it is desirable that the platelet activator 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 the main component, and a saccharide or its salt having a viscosity-average molecular weight of not more than 3,000 as the secondary component. In this specification, the term "main component" refers to the component with the highest weight ratio among the components of the platelet activator. Components other than the main component in the platelet activator are secondary components.

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. The polymers in the platelet activator of the present invention, particularly the hydrophilic groups in the polymers, are presumed to be involved in the vitrification of platelets during freezing, the replacement of water surrounding 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.

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

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

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 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, and a saccharide or its salt having a viscosity-average molecular weight of 3,000 or less.

Preferably, the platelet activator is a monomer having a hydrophilic group that is at least one selected from the group consisting of a hydroxyl group and a carboxylic acid group and salts thereof.

Preferred platelet activators are those 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.

Preferably, the platelet activator is an alternating copolymer of the hydrophilic group-containing monomer and the nitrogen-containing monomer.

Preferably, the platelet activator is one in which the monomer having a hydrophilic group is a monomer having a hydroxyl group substituted at the equatorial position.

Platelet activators containing a polymer or its salt having a viscosity average molecular weight of 5,000 or more are preferred.

Preferably, the platelet activator is a polymer containing multiple sugar residues.

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

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.

Preferred platelet activators are those in which the saccharide is glucose, glucuronic acid, or N-acetylglucosamine.

It is preferable that the polymer or its salt in the platelet activator is present in an amount ranging from 0.1 w/v% to 50 w/v% of the platelet activator.

It is preferable that the sugar or its salt in the platelet activator is present in an amount ranging from 0.1 w/v% to 10 w/v% of the platelet activator.

The present invention also relates to a method for activating a biological sample during cryopreservation, in which the activation is achieved by using a cryoprotectant during cryopreservation.

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.

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

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

The present invention also relates to the 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.

It is preferred that the polymer or its salt and the saccharide or its salt are used as cryoprotectants.

The "viscosity average molecular weight" of the polymer or saccharide used in the present invention is determined using the following method and formula.

Intrinsic viscosity measurement:
(1) A predetermined amount of NaCl is dissolved 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) The stock solution at 30°C is diluted 5/4, 5/3, and 5/2 times with the standard solution at 30°C.
(4) Measure the viscosity of the standard solution, the undiluted solution, and the diluted solution at 30°C. An E-type viscometer is used for the viscosity measurement.
(5) The viscosity of the undiluted solution and the diluted solution is divided by the viscosity of the standard solution to obtain the relative viscosity (η _r ), and the reduced viscosity is calculated based on the following formula.
Here, η _sp is the reduced viscosity of the polymer or sugar [mL/g], η _r is the relative viscosity of the polymer or sugar [-], and C is the concentration of the polymer or sugar [g/mL].
(6) The relationship between the concentration of the polymer or sugar and the reduced viscosity of the polymer or sugar is plotted, and an approximate straight line is drawn. The intercept of the approximate straight line (polymer or sugar concentration = 0) is taken as the limiting viscosity.

Viscosity average molecular weight:
The viscosity average molecular weight is calculated from the intrinsic viscosity.
The viscosity average molecular weight M is calculated from the intrinsic viscosity obtained by measurement and the values of K and α published in literature, etc., using the Mark Hoeing-Sakurada formula described above.

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.

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

In the case of monosaccharides, disaccharides, and compounds that are considered to be monomolecular, the molecular weight is clearly determined from the structural formula, so in this invention, the molecular weight determined from the structural formula is treated as a hypothetical viscosity average molecular weight.

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 sugar 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. It is particularly preferable for the solvent to be an isotonic solution in which the salt concentration, sugar concentration, etc. are adjusted with sodium ions, potassium ions, calcium ions, etc. so that the osmotic pressure is approximately the same as that of body fluids or cellular fluids. Specific examples include water, saline, buffered saline 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 animal cell culture media 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.

The solvent may also contain calcium chloride, magnesium chloride, magnesium sulfate, potassium chloride, potassium dihydrogen phosphate, sodium bicarbonate, disodium hydrogen phosphate, glucose, sodium chloride, an amino acid, or the like. Proline is preferably selected as the amino acid.

The platelet activator of the present invention prevents damage to platelet membranes during cryopreservation by providing solvent vitrification capabilities through the polymeric component with a viscosity-average molecular weight of more than 3000 and not more than 500,000, and inhibiting ice crystal formation and growth near the platelet membrane through the low-molecular-weight sugars with a viscosity-average molecular weight of not more than 3000, thereby protecting the cells. Therefore, after cryopreservation with the platelet activator of the present invention, platelets can maintain their morphology and activation after thawing, making them suitable for advanced cell therapy. That is, platelet activation is achieved during cryopreservation through the action of the polymeric component with a viscosity-average molecular weight of more than 3000 and not more than 500,000 and the low-molecular-weight sugars with a viscosity-average molecular weight of not more than 3000. As a result, platelets cryopreserved with the platelet activator of the present invention and platelets after thawing are activated platelets suitable for use in regenerative medicine.

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 materials containing activated platelets that have been cryopreserved using the platelet activator can be directly applied to cell therapy after thawing. It is possible for the cryopreserved materials to contain low concentrations of chemicals that do not impair the function of activated platelets or their efficacy as cell therapy agents.

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

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.

Furthermore, in the present invention, platelets are cryopreserved and activated with a cryoprotectant at the same time, so there is no need to add a platelet activator to the frozen and thawed platelets for platelet activation. This is thought to reduce the possibility that proteins such as biologically active growth factors will be degraded by the platelet activator. For example, the bovine protein of thrombin used in platelet activation may be a source of pathogens or may cause allergic reactions, but such substances are not necessary for the platelet activation of the present invention.

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. Even during storage of frozen cryopreserved materials, low temperatures such as those achieved by using liquid nitrogen are not required. 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 needed. In other words, 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 material, which can significantly improve the effectiveness of treatment.

Furthermore, such platelets and released intracellular vesicles may be cryopreserved/activated and then provided to a medical institution. In this case, the platelets and released intracellular vesicles provided may be in a frozen state, or in a freeze-dried state in which the solvent is sublimated and removed from the frozen state to produce a powder.

FIG. 1 shows the activation of platelets that have been cryopreserved and then frozen and thawed using the platelet activator of the present invention.

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 polymer of the present invention contains a monomer having a hydrophilic group as a repeating unit.

The viscosity-average molecular weight of the polymer or salt thereof of the present invention is desirably 400,000 or less, and particularly 200,000 or less. This is 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.

The "viscosity average molecular weight" of the polymer or saccharide of the present invention refers to a value calculated using the following method and formula:

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) A predetermined amount of NaCl is dissolved in ion-exchanged water at 30°C to prepare a 0.2 M NaCl solution (standard solution).
(2) A polymer or sugar sample is dissolved in a standard solution at 30°C to prepare a stock solution. If the polymer or sugar sample is obtained in solution, the solid portion obtained by removing the solvent from the solution is used as the polymer or sugar sample. In the case of a mixed sample of polymers and sugars, a mixed sample containing multiple polymers, or a mixed sample containing multiple sugars, each substance is separated and fractionated, and the solvent is removed from each substance to obtain the polymer or sugar sample. Furthermore, if the polymers and/or sugars are unknown, the polymers and/or sugars are identified using HPLC, LC-MS, LC-IR, or other methods. If multiple unknown polymers and/or sugars are contained, each component is separated and fractionated, and the substance of each polymer and/or sugar is identified using HPLC, LC-MS, LC-IR, or other methods, and the viscosity-average molecular weight is calculated as described below. Even if the polymer or sugar contains impurities, as long as they do not affect the viscosity (e.g., impurities such as metal salts), the mixture is 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) The stock solution at 30°C is diluted 5/4, 5/3, and 5/2 times with the standard solution at 30°C.
(4) Measure the viscosity of the standard solution, the undiluted solution, and the diluted solution at 30°C. An E-type viscometer is used for the viscosity measurement.
(5) The viscosity of the undiluted solution and the diluted solution is divided by the viscosity of the standard solution to obtain the relative viscosity (η _r ), and the reduced viscosity is calculated based on the following formula.
Here, η _sp is the reduced viscosity of the polymer or sugar [mL/g], η _r is the relative viscosity of the polymer or sugar [-], and C is the concentration of the polymer or sugar [g/mL].
(6) The relationship between the concentration of the polymer or sugar and the reduced viscosity of the polymer or sugar is plotted, and an approximate straight line is drawn. The intercept of the approximate straight line (polymer or sugar concentration = 0) is taken as the limiting viscosity.
Viscosity average molecular weight:
The viscosity average molecular weight is calculated from the intrinsic viscosity.
The viscosity-average molecular weight M is calculated from the intrinsic viscosity determined by measurement and the K and α values published in literature, etc., in the Mark Hoeing-Sakurada formula. In the case of hyaluronic acid, K = 3.6 × 10 ^-4 and α = 0.78 are substituted to calculate the viscosity-average molecular weight M. For pullulan and gelatin used in the examples, K = 9 × 10 ^-4 and α = 0.5 are used from literature values; for dextran, K = 6.3 × 10 ^-8 and α = 1.4; for chondroitin sulfate, K = 5.8 × 10 ^-4 and α = 0.74; and for carboxypolylysine, K = 2.78 × 10 ^-5 and α = 0.87 are used.

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.

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 can be clearly determined from the structural formula, and therefore the molecular weight determined from the structural formula is treated as a hypothetical viscosity average molecular weight.

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 increased concentration of solutes (cryoprotectants) or the increased cooling rate required 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 intracellular osmotic pressure difference 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 frozen storage is thawed, it is thought that there is little damage to the platelets contained in the frozen storage when thawed.

The viscosity-average molecular weight of the polymer of the present invention is greater than 3,000 and less than 500,000. A viscosity-average molecular weight of this level can stabilize the amorphous, vitreous state in the frozen state in 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. If the viscosity-average molecular weight of the polymer is 3,000 or less, vitrification may not occur well. Furthermore, if the viscosity-average molecular weight of the polymer is greater than 500,000, the viscosity may increase significantly, resulting in reduced solubility in solvents and foaming of the solution before cryopreservation, which may result in poor handling. For example, 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 allows the viscosity of the platelet activator to be adjusted low, making it easier to handle.

The polymer of the present invention is a polymer containing, as a repeating unit, a monomer having a hydrophilic group. Examples of hydrophilic groups include hydroxyl groups and carboxylic acid groups and salts thereof. The polymer of the present invention may also contain, as a repeating unit, 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 thought to enable the water solvent to be more effectively trapped within the matrix formed by the polymer chains during freezing.

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 in which sugar residues are linked as repeating units via glycosidic bonds, or a polymer containing derivatives thereof. Examples of sugar residues include, but are not limited to, monosaccharides, or monosaccharides in which the hydroxyl and/or hydroxymethyl groups of a monosaccharide 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.

Monosaccharides include trioses, tetroses, pentoses, hexoses, and heptoses. For example, pentoses include ribose, arabinose, xylose, lyxose, xylulose, ribulose, and deoxyribose. Hexoses include glucose, mannose, galactose, fructose, sorbose, tagatose, fucose, fuculose, and rhamnose.

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.

For example, the polymer of the present invention is a polymer containing the above-mentioned monosaccharides as a repeating unit. 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 a repeating unit. 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, the polymer of the present invention may be, for example, a glycosaminoglycan. It may also 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.

The polymers used in the present invention may be naturally occurring, chemically synthesized, or commercially available. Naturally occurring or commercially available polymer compounds 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 chemically synthesized. For example, preferably, the monomers contained in the polymers of the present invention are biological components. These monomers have the advantage of low cytotoxicity and the ability to be directly cryopreserved and used in cell preparations.

The hydrophilic groups of the polymer of the present invention are preferably unmodified or, if modified, account for 50% or less of the total number of hydrophilic groups, i.e., no substituents are introduced into the polymer chain or, if introduced, account for 50% or less of the total number of hydrophilic groups. It is believed that the hydrophilic groups of the polymer, particularly OH groups, _NH2 groups, 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.

Furthermore, the hydrophilic groups of the polymers of the present invention are thought to hold low-molecular-weight sugars through 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 NH2 _groups with carboxylic acids or the like may not be desirable.

The platelet activator of the present invention contains a saccharide or salt thereof having a viscosity-average molecular weight of 3000 or less. This saccharide replaces water molecules near the cell membrane of platelet cells, 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. In other words, the saccharide or salt thereof 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 3000 or less, preferably 2000 or less, and more preferably 1000 or less.

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.

Preferably, the saccharide is glucose or a cleavage product of hyaluronic acid. Therefore, preferably, the saccharide of the present invention is glucose, glucuronic acid, or N-acetylglucosamine, or a disaccharide or oligosaccharide composed thereof. Preferably, the saccharide may be glucuronic acid or a modified compound thereof, or a disaccharide or oligosaccharide thereof.

As used herein, the term "cleavage product" refers to a compound with a smaller molecular weight than the original polymer, which is believed to be obtained when a polymer is subjected to hydrolysis, enzymatic treatment, subcritical treatment, or other treatment. That is, the polymer of the present invention may be a polymer with 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 with 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.

"Subcritical processing" refers to contacting the raw material to be extracted with a subcritical fluid (extraction solvent) that has been brought to a subcritical state under specified temperature and pressure conditions. For example, water is neither a liquid nor a gas when its pressure reaches 22.12 MPa or higher and its temperature reaches 374.15°C or higher. This point is called the critical point of water, and hot water at temperatures and pressures lower than or equal to 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. The subcritical processing pressure can be equal to or higher than the saturated vapor pressure at each temperature, but can be, for example, 0.5 MPa or higher and 25 MPa or lower. After subcritical processing, components with molecular weights below a specified level are separated and recovered and can be used as the cleavage product of 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.

The polymer and saccharide of the present invention may be obtained simultaneously by a single subcritical treatment. That is, the polymer and saccharide of the present invention may be a subcritical treatment product 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 in viscosity average molecular weight, and a second molecular weight distribution in a molecular weight range of not more than 3,000 in viscosity average molecular weight.

Salts of the polymers or saccharides 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 that can be selected include sodium, potassium, and calcium. Examples of halogens that can be used include chlorine and bromine.

The platelet activator of the present invention includes a polymer or its salt having a viscosity-average molecular weight greater than 3,000 and less 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 of less than 3,000. These polymers are cryoprotectants for cryopreservation. Specifically, the platelet activator of the present invention contains a cryoprotectant that combines a polymer that traps water molecules with polymer chains during freezing, preventing ice crystals from forming in the solvent and vitrifying the sample, thereby preventing rupture of biological samples such as cells due to ice crystal formation, with 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. This cryoprotectant of the present invention effectively inhibits ice crystal formation during freezing and recrystallization during thawing, thereby providing the resulting cryopreserved product with a high cryopreservation effect not attainable 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 frozen samples, the activated state of the platelets is maintained, and the platelets retain their biological activity and therapeutic effects as activated platelets.

In the platelet activator of the present invention, the polymer or its salt is present in the platelet activator in an amount ranging from approximately 0.1 w/v% to 50 w/v% of the platelet activator. If the amount is less than 0.1 w/v%, the solvent portion of the frozen cryopreserved product may not be vitrified satisfactorily. Furthermore, if the amount is more than 50 w/v%, the viscosity of the solution containing the polymer or its salt may become too high, 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. Furthermore, 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.

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

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 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) 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).

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. to approximately the same as the osmotic pressure of body fluids or cellular fluids. Specific examples include, but are not limited to, water, saline, buffered saline solutions 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, as long as the effects of the present invention are not impaired, the solvent may contain other optional components such as isotonic agents, chelating agents, solubilizers, pH adjusters, and additives commonly used in cell culture media.

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 sugars 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, cell membrane-impermeable cryoprotectants may be used as cryoprotective support substances, such as sugars and dextran. Examples of sugars include dextrose, mannose, galactose, fructose, raffinose, lactose, sucrose, maltose, glucose, sorbitol, mannitol, and trehalose. Such cryoprotective support substances can 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.

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

For example, the solvent for the platelet activator of the present invention may be a 5% glucose aqueous solution. The solvent may also 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.

Cryopreservation using the platelet activator of the present invention results in minimal cellular damage to frozen platelets. This is because a biological sample is effectively frozen to produce 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 the high cooling rate 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 desirably -70°C or below, and preferably -80°C or below. The lower limit is desirably -196°C or higher, and preferably -150°C or higher.

The storage period of the frozen preserved material containing a biological sample of the present invention is not particularly limited, but may be, for example, one week or more, two weeks or more, three weeks or more, four weeks or more, two months or more, three months or more, four months or more, five months or more, six months or more, one year or more, or longer. Even after such long-term storage in a frozen state, platelets remain activated and maintain a high survival rate after thawing.

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 repeating unit containing a monomer having a hydrophilic group, 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 in conjunction 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.

As a polymer of the present invention having a viscosity-average molecular weight of greater than 3,000 and less than 500,000, and containing 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 cryopreserved material. For example, a preferred polymer of the present invention is hyaluronic acid. In particular, it is hyaluronic acid with a viscosity-average molecular weight of less than 400,000, preferably less than 200,000. More particularly, it includes hyaluronic acid with a viscosity-average molecular weight of greater than 3,000, more preferably greater than 5,000, and less than 60,000, more preferably less than 20,000.

The present invention will be specifically explained based on examples, but the present invention is not limited to these.

<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 treatment temperature of 175°C, a treatment pressure of 0.89MPa, and a treatment time of 3 minutes.Then, the subcritical treatment product was dried by freeze-drying or spray-drying.As a result, a mixture of high molecular weight hyaluronic acid with an intrinsic viscosity of 0.49dL/g and a viscosity-average molecular weight of 10,000 and low molecular weight hyaluronic acid with an intrinsic viscosity of 0.08dL/g and a viscosity-average molecular weight of 1,000 was obtained, that is, the high molecular weight and low molecular weight sugars used as cryoprotectants in the present invention were obtained.

<Preparation of platelets>
Platelets isolated from fresh blood collected from C57BL/6 mice (15 weeks old, male) were used. 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, 20°C, and 5 minutes to separate blood cells and platelet-rich plasma (PRP). The PRP was collected and centrifuged (1000 rpm, 20°C, and 5 minutes) to remove as many blood cells as possible from the PRP. Next, the PRP was centrifuged at 3600 rpm, 20°C, and 5 minutes to separate platelets and platelet-poor plasma (PPP), and only the platelets were collected.

<Preparing the platelet sample>
The separated and collected platelets were sedimented by centrifugation and then resuspended in Tyrode buffer (composition: NaCl 8 g/L, KCl 0.2 g/L, CaCl _0.2 g/L, MgCl _{6H O} 0.1 g/L, NaH PO _{4 H} O _0.05 g/L, glucose 1 g/L, NaHCO ₁ g/L) at a concentration of 2 × ¹⁰ cells/mL to prepare a platelet solution.

Activation of Unfrozen Platelets and Measurement of Activation
Comparative Example 1
The platelet solution obtained above was collected so that the platelet concentration was 1 x ¹⁰⁷ platelets, and diluted with an equal volume of Tyrode's buffer. As a negative control, 20 µL of Tyrode's buffer was added.

After replacing the medium with Tyrode's buffer containing 1 mM _CaCl , 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-labeled antibodies (anti-CD41/61) and fluorescent dye FITC-labeled antibodies (anti-P-selectin/CD62-P) (EMFRET Analytics GmbH & Co. KG, catalog number: D200) were used.

Comparative Example 2
As in Comparative Example 1, a platelet solution was collected to a concentration of 1 x ¹⁰ platelets and diluted with an equal volume of Tyrode buffer. 20 μL of 1 U/mL thrombin (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to activate the platelets.

CD41/61 and CD62-P were evaluated in the same manner as in Comparative Example 1.

<Measurement of frozen platelet activation>
Example 1
100 g of the cryoprotectant, a mixture of high molecular weight and low molecular weight saccharides, prepared as described above, was added to 1 L 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 wt% of the mixture of high molecular weight and low molecular weight saccharides, with a ratio of high molecular weight hyaluronic acid having a viscosity average molecular weight of 10,000 to low molecular weight hyaluronic acid having a viscosity average molecular weight of 1,000 = 10:1).

Platelet solution was collected to a concentration of 1 x ¹⁰ platelets and diluted with an equal volume of the platelet activator prepared above. The resulting sample was placed in a cryopreservation container (Mr. Frosty) and frozen in a deep freezer at a cooling rate of 1°C/min. The sample was then frozen and stored until use.

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

Comparative Example 3
As in Comparative Example 1, a platelet solution was collected to give a concentration of 1 x ¹⁰ platelets 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.

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

Comparative Example 4
Platelet solution was collected to a concentration of 1 × ¹⁰ platelets 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 an aqueous solution of 5 g of sodium carboxymethylcellulose (molecular weight 760,000) dissolved in 750 mL of distilled water and an aqueous solution of 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 dissolved in 150 mL of distilled water). The resulting sample was placed in a cryopreservation container (Mr. Frosty), frozen at a cooling rate of 1°C/min in a deep freezer, and stored frozen until use.

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

The flow cytometry detection results for Comparative Examples 1 to 4 and Example 1 are shown in Figure 1.

In the results shown in Figure 1, Comparative Examples 1 to 4 and Example 1 respectively represent 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).

As shown in Figure 1, platelets cryopreserved using the platelet activator of the present invention showed enhanced cell surface migration (expression) of CD62-P and CD41/61. High platelet activation was confirmed when platelets were cryopreserved using the platelet activator of the present invention. This degree of activation was higher than the results of unfrozen platelets activated with thrombin (Comparative Example 2) and cryopreserved under conditions that did not include the cryoprotectant of the present invention (Comparative Example 3). Such activation was not confirmed when platelets were cryopreserved using a commercially available cryopreservation solution.

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 using the platelet activator of the present invention simultaneously preserves and activates biological samples. Therefore, from the standpoints of biological activity and simplicity, the platelet activator of the present invention may be advantageously applied to regenerative medicine.

Claims (6)

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;
1. A platelet activator comprising hyaluronic acid or a salt thereof having a viscosity average molecular weight of 3,000 or less,
A platelet activator that activates platelets during cryopreservation. 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. 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. 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. 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. 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.