Method for evaluating platelet function

Abstract

The problem to be addressed by the present invention is to provide a method and the like for evaluating platelet function in a simple manner with a high level of accuracy.　A method for evaluating platelet function according to the present invention comprises a step A for bringing platelets into contact with a solution containing a calcium-containing compound, and following step A, a step B for detecting or quantifying cytokines released by the platelets.

Description

Methods for assessing platelet function

[0001] The present invention relates to a method for evaluating platelet function, etc.

[0002] The medical uses of platelets are mainly for blood transfusion and platelet-rich plasma (hereinafter also referred to as "PRP") therapy. PRP is a highly concentrated form of platelets, one of the three types of blood cells (red blood cells, white blood cells, and platelets) contained in a patient's own blood, obtained by centrifuging the patient's own blood. PRP contains cytokines such as VEGF (Vascular Endothelial Growth Factor), FGF (Fibroblast Growth Factor), TGF (Transforming Growth Factor), EGF (Epidermal Growth Factor), PDGF (Platelet-Derived Growth Factor), IGF (Insulin-like Growth Factor), and HGF (Hepatocyte Growth Factor).

[0003] PRP is a platelet-enriched plasma prepared by centrifuging blood. Platelet-rich plasma is rich in a variety of growth factors, which play an effective role in wound healing and tissue regeneration, making it a promising material in the field of regenerative medicine (see, for example, Patent Document 1). However, platelets have the problem of decreasing in function over time after collection (see, for example, Patent Document 2), and once they become dead cells, they no longer function as platelets.

[0004] Methods using staining substances are known for determining cell viability. For example, trypan blue is taken up by dead cells, passes through the cell membrane, and stains the cytoplasm blue; this property can be used to determine cell viability. However, trypan blue cannot be used to determine viability depending on the type of cell, as it is not taken up by red blood cells. Furthermore, propidium iodide (PI) and 7-amino-actinomycin D (7-ADD) bind to nucleic acids, intercalate into DNA, and cause dead cells to develop a bright color; this property can be used to determine cell viability. However, because platelets do not have a nucleus, they are not stained by PI or 7-ADD. Furthermore, because the permeability of the platelet cell membrane is unique, the presence or absence of staining cannot be clearly confirmed with trypan blue. Thus, existing methods are unable to determine the viability of platelets.

[0005] Known methods for evaluating platelet function include measuring the expression level of P-selectin (CD62P) on the platelet surface (Patent Document 3) and the expression level of PAC-1 (Patent Document 4) by flow cytometry. However, these methods are poorly quantitative and it is difficult to standardize the test methods, so they are only used by a few experts for research purposes.

[0006] JP 2009-195739 A JP 2021-147333 A JP 2012-008044 A JP 2004-117152 A

[0007] An object of the present invention is to provide a method for evaluating platelet function simply and with high accuracy.

[0008] The present inventors have intensively investigated various methods to solve the above-mentioned problems, and have found that the function of platelets can be evaluated by contacting platelets with a solution containing a calcium-containing compound and then detecting or quantifying cytokines released by the platelets, thereby solving the above-mentioned problems and completing the present invention.

[0009] That is, the present invention provides the following inventions, etc.: [1] A method for evaluating platelet function, comprising: Step A of contacting platelets with a solution containing a calcium-containing compound; and Step B of detecting or quantifying cytokines released by the platelets after Step A; [2] The method according to [1] above, wherein the calcium concentration of the solution containing the calcium-containing compound is 0.01 to 100 mM; [3] The method according to [1] or [2] above, wherein the time for contacting platelets with the solution containing the calcium-containing compound is 0.1 minutes to 3 hours; [4] A method according to [1] above, wherein the cytokines are selected from the group consisting of VEGF (Vascular Endothelial Growth Factor), FGF (Fibroblast Growth Factor), TGF (Transforming Growth Factor), EGF (Epidermal Growth Factor), PDGF (Platelet-Derived Growth Factor), IGF (Insulin-like Growth Factor), and HGF (Hepatocyte Growth Factor). [5] The method according to any one of [1] to [4] above, further comprising, after step B, step C of evaluating the platelets as having platelet function when the platelets release cytokines as a result of contacting the platelets with a solution containing a calcium-containing compound; [6] The method according to any one of [1] to [5] above, further comprising, before step A, step P of centrifuging a liquid containing platelets to isolate platelets; [7] The method according to any one of [1] to [6] above, further comprising, after step A and before step B, step Q of centrifuging a liquid containing platelets to isolate platelets;

[0010] According to the present invention, a method for evaluating platelet function simply and with high accuracy can be provided.

[0011] The present invention includes a method for evaluating platelet function (hereinafter also referred to as the "method of the present invention"), which comprises: step A of contacting platelets with a solution containing a calcium-containing compound; and step B of detecting or quantifying cytokines released by the platelets after step A.

[0012] <Method of the Present Invention> The method of the present invention is not particularly limited as long as it is a method for evaluating platelet function, comprising: step A of contacting platelets with a solution containing a calcium-containing compound; and step B of detecting or quantifying cytokines released by the platelets after step A.

[0013] (Step A) Step A is not particularly limited as long as it is a step of bringing platelets into contact with a solution containing a calcium-containing compound. The method of bringing platelets into contact with the solution containing a calcium-containing compound is not particularly limited, and the solution containing the calcium-containing compound may be added to the platelets, the platelets may be added to the solution containing the calcium-containing compound, the calcium-containing compound may be added to a liquid containing platelets, or the calcium-containing compound may be added to a liquid containing platelets.

[0014] (Platelets) As used herein, "platelets" include mammalian platelets, and more specifically, "platelets derived from mammalian blood" and "platelets produced by in vitro induction from mammalian-derived cells or cell lines" are preferred. Among these, "platelets produced by in vitro induction from mammalian-derived cells or cell lines" are more preferred, "platelets produced by in vitro induction from mammalian-derived cell lines" are even more preferred, and "platelets produced by in vitro induction from mammalian adipose tissue-derived mesenchymal stem cell lines" are even more preferred. Platelets produced from ASCL, as described below, are even more preferred due to their greater effectiveness in wound healing and tissue regeneration, and "ASCL-PLC" owned or produced by AdipoSeeds, Inc. is particularly preferred. Mammalian platelets can be obtained from a mammalian blood sample. Furthermore, the platelets used herein may be isolated or contained in a liquid (e.g., a blood sample).

[0015] As used herein, the term "mammal" refers to, for example, humans and non-human mammals, with humans being preferred. Examples of non-human mammals include mice, rats, guinea pigs, dogs, cats, monkeys, rabbits, sheep, horses, and pigs.

[0016] The "blood sample" is not particularly limited as long as it is a blood sample containing platelets, and examples thereof include whole blood. A blood sample can be collected from a mammal by a conventional method.

[0017] The above-mentioned "platelets produced by in vitro induction from mammalian-derived cells or cell lines" are not particularly limited, as long as they are platelets produced by in vitro induction from mammalian-derived cells or cell lines. Such mammalian-derived cells and cell lines are not particularly limited, as long as they are cells or cell lines that can be induced to become platelets in vitro (hereinafter also referred to as "starting cells, etc."). From the viewpoints of ease of availability and the stability of the quality of the platelets obtained, mammalian-derived cell lines are preferred. Examples of mammalian-derived cells include "mammalian hematopoietic stem cells" and "one or more cells selected from a group of stromal vascular cells comprising mesenchymal stem cells, preadipocytes, and stromal cells of mammalian adipose tissue." From the viewpoint of ease of availability, etc., "one or more cells selected from a group of stromal vascular cells comprising mesenchymal stem cells, preadipocytes, and stromal cells of mammalian adipose tissue" are preferred. Furthermore, examples of the aforementioned mammal-derived cells and cell lines include "mammalian hematopoietic stem cell lines" and "cell lines of one or more cells selected from a stromal vascular cell group comprising mesenchymal stem cells, preadipocytes, and stromal cells of mammalian adipose tissue." From the viewpoint of high cytokine release ability, etc., preferred are "cell lines of one or more cells selected from a stromal vascular cell group comprising mesenchymal stem cells, preadipocytes, and stromal cells of mammalian adipose tissue" (i.e., Adipose-derived Mesenchymal Stem / stromal Cell Lines: ASCLs) (in this specification, ASCLs with no particular owner specified refer to such cell lines), and particularly preferred are "ASCLs" owned by AdipoSeeds, Inc. (i.e., ASCLs that are adipose tissue-derived mesenchymal stem cell lines). A preferred example of a "method for producing a cell line (preferably a mesenchymal stem cell line) from one or more types of cells selected from a group of stromal vascular cells including mesenchymal stem cells, preadipocytes, and stromal cells of mammalian adipose tissue" is the method described in Japanese Patent No. 6714932, and a "method for producing a cell line by inducing mammal-derived cells or cell lines to become platelets in vitro" is as described below in "(A method for producing a cell line by inducing mammal-derived cells or cell lines to become platelets in vitro)."

[0018] (Solution Containing a Calcium-Containing Compound) The "solution containing a calcium-containing compound" in the present invention is not particularly limited as long as it is a solution containing a calcium-containing compound, and preferred examples include an aqueous solution containing a calcium-containing compound, and more preferred examples include phosphate buffered saline containing a calcium-containing compound. The "calcium-containing compound" includes water-soluble calcium-containing compounds, and preferred examples include water-soluble calcium salts. Examples of the "water-soluble calcium salt" include one or more selected from the group consisting of calcium chloride, calcium hydroxide, calcium acetate, calcium carbonate, calcium bicarbonate, calcium chlorate, calcium perchlorate, calcium sulfate, calcium nitrate, calcium nitrite, calcium lactate, calcium glubionate, calcium gluceptate, and calcium gluconate, and preferred examples include calcium chloride.

[0019] The calcium concentration in the above-mentioned "solution containing a calcium-containing compound" is not particularly limited as long as the effects of the present invention can be obtained, but examples thereof include 0.01 to 100 mM, preferably 0.5 to 50 mM, and more preferably 5 to 20 mM.

[0020] In the above step A, the time for which platelets are brought into contact with the solution containing a calcium-containing compound is not particularly limited as long as the effects of the present invention are obtained, but may be, for example, 0.1 minute to 3 hours, preferably 1 to 60 minutes, preferably 3 to 40 minutes, and more preferably 10 to 20 minutes.

[0021] (Step P) The method of the present invention does not necessarily have to further include, as an optional step, step P of isolating platelets by centrifuging a liquid containing platelets before step A. However, from the viewpoint of evaluating platelet function with higher accuracy, it is preferable that the method of the present invention further includes step P. If the method of the present invention further includes step P, cytokines released by platelets can be removed by performing step P before performing step A of contacting the platelets with a solution containing a calcium-containing compound. As a result, it is preferable that the cytokines detected or quantified in step B can be determined to be cytokines released after the platelets were contacted with the solution containing a calcium-containing compound.

[0022] In step P, "centrifugally separating a platelet-containing liquid to isolate platelets" means separating the platelet-containing liquid from the liquid by centrifuging the platelet-containing liquid. The method for isolating platelets by centrifuging a platelet-containing liquid in step P is not particularly limited, as long as it is a method for recovering platelets by centrifugation. Centrifugation conditions include 400 to 2000 G (preferably 600 to 1800 G, more preferably 800 to 1600 G, even more preferably 900 to 1500 G, or 1000 to 1400 G) for 1 to 25 minutes (preferably 3 to 20 minutes, more preferably 4 to 18 minutes, and even more preferably 5 to 15 minutes). By precipitating the platelets through centrifugation and removing the supernatant, platelets can be isolated. Centrifugation can be performed using a commercially available centrifuge or the like.

[0023] (Step B) Step B is not particularly limited as long as it is a step of detecting or quantifying cytokines released by the platelets after Step A, but a preferred embodiment is Step B', in which cytokines released by the platelets are detected or quantified after contacting the platelets with a solution containing a calcium-containing compound. A preferred example of a method for detecting or quantifying cytokines in Step B' is a method in which, before Step A, in which platelets are contacted with a solution containing a calcium-containing compound, a liquid containing platelets is centrifuged to isolate the platelets, thereby carrying out Step P.

[0024] The "cytokine" used herein is not particularly limited as long as it is a cytokine released by platelets, and examples thereof include one or more cytokines selected from the group consisting of VEGF (Vascular Endothelial Growth Factor), FGF (Fibroblast Growth Factor), TGF (Transforming Growth Factor), EGF (Epidermal Growth Factor), PDGF (Platelet-Derived Growth Factor), IGF (Insulin-like Growth Factor), and HGF (Hepatocyte Growth Factor); preferred examples include one or more cytokines selected from the group consisting of VEGF, FGF (preferably bFGF), and TGF (preferably TGF-β); and from the viewpoint of simplicity, more preferred example includes one cytokine selected from VEGF, FGF (preferably bFGF), and TGF (preferably TGF-β).

[0025] The method for detecting or quantifying cytokines is not particularly limited as long as it is capable of detecting or quantifying cytokines, and examples thereof include enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunohistochemistry, and radioimmunoassay (RIA). From the viewpoints of simplicity and speed, ELISA is preferred. Furthermore, the antibodies used in these methods can be commercially available or labeled monoclonal or polyclonal antibodies appropriately prepared by known antibody preparation methods. The label can be an enzyme, a radioisotope, a fluorescent dye, or the like. The enzyme is not particularly limited as long as it satisfies the following conditions: it has a high metabolic turnover number, is stable when bound to the antibody, and specifically colors the substrate. Enzymes used in conventional enzyme immunoassays (EIA), such as peroxidase, β-galactosidase, alkaline phosphatase, glucose oxidase, acetylcholinesterase, glucose-6-phosphate dehydrogenase, and malate dehydrogenase, can also be used. Enzyme inhibitors, coenzymes, and the like can also be used. The binding of these enzymes to antibodies can be carried out by known methods using cross-linking agents such as maleimide compounds. Known substances can be used as substrates depending on the type of enzyme used. For example, when peroxidase is used as the enzyme, 3,3',5,5'-tetramethylbenzidine can be used, and when alkaline phosphatase is used as the enzyme, paranitrophenol or the like can be used. As radioisotopes, １２５ I and ３Fluorescent dyes used in conventional RIAs, such as Fluorescein Isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC), can be used. Fluorescent dyes used in conventional fluorescent antibody assays, such as fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC), can be used. When using an enzyme, a substrate that decomposes and develops color through enzymatic action is added, and the amount of substrate decomposition is optically measured to determine the enzyme activity, which is converted into the amount of bound antibody, and the amount of antibody is calculated by comparing it with a standard value. When using a radioisotope, the radiation dose emitted by the radioisotope is measured using a scintillation counter or the like. When using a fluorescent dye, the amount of fluorescence can be measured using a measuring device combined with a fluorescence microscope. Measurements can also be performed using a flow cytometer. Furthermore, a sandwich method using a primary antibody and a labeled secondary antibody (called "ELISA" when an enzyme is used as the label) is also preferably used. For immunohistochemical measurements, a commercially available cytokine antibody array kit can be used to comprehensively confirm the production of a wide variety of cytokines. Cytokines released by platelets can be detected or quantified using the above methods.

[0026] (Step Q) The method of the present invention does not necessarily have to include, but preferably includes, an optional step, step Q of centrifuging a platelet-containing liquid to isolate platelets after step A and before step B. The method of the present invention preferably further includes step Q, since it allows platelet function to be evaluated with higher accuracy. When the method of the present invention further includes step Q, it is preferable to detect or quantify cytokines in the supernatant obtained by centrifugation in step Q as the detection or quantification of cytokines in step B.

[0027] In step Q, "centrifugally separating the platelet-containing liquid to isolate the platelets" means separating the platelet-containing liquid from the liquid by centrifuging the platelet-containing liquid. The method for isolating the platelets by centrifuging the platelet-containing liquid in step Q is not particularly limited, as long as it involves recovering platelets by centrifugation. Examples of centrifugation conditions include 400 to 2000 G (preferably 600 to 1800 G, more preferably 800 to 1600 G, even more preferably 900 to 1500 G, or 1000 to 1400 G) for 1 to 25 minutes (preferably 3 to 20 minutes, more preferably 4 to 18 minutes, and even more preferably 5 to 15 minutes). By precipitating the platelets through centrifugation and removing the supernatant, platelets can be isolated. When the method of the present invention includes steps P and Q, the centrifugation conditions in step P and step Q may be the same or different.

[0028] (Optional Steps) The method of the present invention is not particularly limited as long as it includes the above-mentioned steps A and B, but may also include other optional steps, for example, it may further include one or more (preferably three) steps selected from the group consisting of the below-described step C, the above-described step P, and the above-described step Q, and it is preferable that the method of the present invention further includes one or more (preferably three) steps selected from the group consisting of step C, step P, and step Q. Note that when the method of the present invention includes step C, step C can be included after step B.

[0029] (Step C) Step C is not particularly limited as long as it is a step of evaluating the platelets as having platelet function when the platelets release cytokines as a result of contacting the platelets with a solution containing a calcium-containing compound.

[0030] Whether or not cytokines have been released from platelets as a result of contacting the platelets with a solution containing a calcium-containing compound can be confirmed or determined by those skilled in the art from the results of detecting or quantifying the cytokines released by the platelets in step B. For example, in the method of the present invention, step P of isolating platelets by centrifuging a platelet-containing liquid is carried out before step A of contacting the platelets with a solution containing a calcium-containing compound, the cytokines detected or quantified in step B can be determined to be cytokines released after the platelets were contacted with the solution containing a calcium-containing compound. On the other hand, in the method of the present invention, which does not further include step P and, for example, is a method in which a calcium-containing compound is added to a platelet-containing liquid without isolating the platelets, it can be determined that cytokines have been released from the platelets as a result of contacting the platelets with a calcium-containing compound when, for example, the cytokine concentration after a certain period of time has elapsed after the addition of the calcium-containing compound is increased compared to the cytokine concentration before the addition of the calcium-containing compound. Even if the cytokine concentration does not increase after a certain period of time has elapsed since the addition of the calcium-containing compound compared to the cytokine concentration before the addition of the calcium-containing compound, if the cytokine amount is calculated taking into account the dilution rate of the liquid used for detection or measurement in step B and it can be determined that "contact of platelets with the calcium-containing compound caused the platelets to release cytokines," then this can be determined.

[0031] More specifically, examples of "when platelets have been brought into contact with a solution containing a calcium-containing compound, resulting in the release of cytokines from the platelets" in step C include when, in the method of the present invention comprising steps P, A, Q, and B, at least one cytokine (e.g., VEGF) is present at a concentration equal to or higher than the detection limit, preferably 10 pg / mL or more, 20 pg / mL or more, 30 pg / mL or more, 40 pg / mL or more, 50 pg / mL or more, more preferably 60 pg / mL or more, 70 pg / mL or more, 80 pg / mL or more, 90 pg / mL or more, 100 pg / mL or more, even more preferably 110 pg / mL or more, 120 pg / mL or more, 130 pg / mL or more, 140 pg / mL or more, 150 pg / mL or more, more preferably 160 pg / mL or more, 170 pg / mL or more, 180 pg / mL or more, 190 pg / mL or more, or 200 pg / mL or more.

[0032] (Function of Platelets) In the present specification, the "function of platelets" is not particularly limited as long as it is a function of platelets, but preferred examples include the ability to release cytokines, and more preferred examples include the ability to release one or more cytokines selected from the group consisting of VEGF, FGF, TGF, EGF, PDGF, IGF, and HGF, the ability to release one or more cytokines selected from the group consisting of VEGF, FGF (preferably bFGF), and TGF (preferably TGF-β), and the ability to release one cytokine selected from VEGF, FGF (preferably bFGF), and TGF (preferably TGF-β).

[0033] (Method for producing platelets by inducing mammalian-derived cells or cell lines in vitro) The method for producing platelets by inducing mammalian-derived cells or cell lines (starting cells, etc.) in vitro (hereinafter also referred to as "platelet production method") is not particularly limited, and any known method can be used. Specific examples of such methods include a method comprising a step R of culturing starting cells, etc. in a differentiation-inducing medium for megakaryocytic cells containing iron ions and an iron transporter, and collecting platelets from the culture.

[0034] The "culture medium for inducing differentiation into megakaryocytic cells, containing iron ions and an iron transporter" in step R (hereinafter also referred to as "culture medium in step R") is a culture medium in which iron ions and an iron transporter are added to a basal culture medium for mesenchymal cell culture. The addition of iron ions and an iron transporter has the effect of inducing differentiation of starter cells, etc. into platelets.

[0035] The iron ion may be either iron (II) ion or iron (III) ion, with iron (III) ion being preferred. A method for incorporating iron ions into the culture solution in step R includes adding one or more iron salts selected from the group consisting of inorganic and organic iron salts to a culture solution for inducing differentiation into megakaryocytic cells. Such iron salts may be organic or inorganic salts, and examples of the inorganic salts include iron chloride (II), iron chloride (III), iron oxide (II), iron oxide (III), iron nitrate (II), iron nitrate (III), iron sulfate (II), iron sulfate (III), ammonium iron sulfate (II), ammonium iron sulfate (III), iron pyrophosphate (II), iron pyrophosphate (III), iron sulfide (II), iron sulfide (III), iron hydroxide (II), and iron hydroxide (III). Examples of the organic salts include iron acetate (II), iron acetate (III), hydroxydiacetoxyiron (III), iron citrate (II), iron citrate (III), sodium iron citrate (III), ammonium iron citrate (III), iron benzoate (II), iron benzoate (III), carbonate, and the like. Examples of suitable iron salts include iron(II), iron(III), formate(II), formate(III), oxalate(II), oxalate(III), fumarate(II), fumarate(III), succinate(II), succinate(III), gluconate(II), gluconate(III), lactate(II), lactate(III), maleate(II), maleate(III), sodium iron(III) diethylenetriaminepentaacetate, ammonium iron(III) diethylenetriaminepentaacetate, sodium iron(III) ethylenediaminetetraacetate, ammonium iron(III) ethylenediaminetetraacetate, sodium iron(III) dicarboxymethylglutamate, and ammonium iron(III) dicarboxymethylglutamate. These iron salts may be used alone or in combination of two or more. Commercially available iron salts may be used.

[0036] The iron transporter binds to iron ions contained in the culture medium in step R, providing the starter cells with the ability to take up iron ions from the culture medium. Furthermore, if an iron transporter bound to iron ions is used, it can also function as an iron source. Iron transporters are sometimes called apo-type when not bound to iron, holo-type when bound to iron, and sidero-type when bound to an amount of iron intermediate between the apo-type and holo-type. Examples of the iron transporter include proteins that bind to iron and are taken up into cells (see, for example, JP 08-029429 A, JP 2005-517042 A, JP 2004-505932 A, and JP 2007-508026 A). Examples of apo-type or equivalent iron transporters include apotransferrin (apocerotransferrin), apolactoferrin, apoovotransferrin, apomelanotransferrin, apoferritin, and protoporphyrin IX, with apotransferrin being preferred. The biological species from which such iron transporters are derived is preferably the same as the biological species from which the starter cells or the like used together are derived.

[0037] As the iron ion and iron transporter in the present invention, a complex formed by binding of an iron ion and an iron transporter (iron ion-iron transporter complex) can be preferably used. Examples of such iron ion-iron transporter complexes include holotransferrin (iron-binding transferrin) in which apotransferrin is bound to an iron ion, hololactoferrin (iron-binding lactoferrin) in which apolactoferrin is bound to an iron ion, holovotransferrin (iron-binding ovotransferrin) in which apoovotransferrin is bound to an iron ion, holomelanotransferrin (iron-binding melanotransferrin) in which apomelanotransferrin is bound to an iron ion, holoferritin (iron-binding ferritin) in which apoferritin is bound to an iron ion, and heme in which protoporphyrin IX is bound to iron. Among these, iron-binding transferrin is particularly preferred. The above-mentioned holo-type iron transporters that are bound to iron ions, apo-type iron transporters that are not bound to iron ions, and sidero-type iron transporters that are bound to an amount of iron at an intermediate level between the apo-type and holo-type can be commercially available.

[0038] The bond between the iron ion and the iron transporter in the iron ion-iron transporter complex is not particularly limited, and may be a non-covalent bond such as a coordinate bond, ionic bond, hydrogen bond, metallic bond, or van der Waals force, or a covalent bond, but a coordinate bond is preferred because it provides an appropriate degree of bond and is suitable for transporting iron ions into start cells, etc. For convenience, the iron ion-iron transporter complex of the present invention also includes complexes in which iron is not in an ionic state but which can release iron ions when the complex is taken up into start cells, etc.

[0039] The iron ion concentration in the culture medium in step R is not particularly limited, as long as the platelets of the present invention can be produced by culturing starter cells or the like in the culture medium used in the present invention having such an iron concentration. Examples of the iron ion concentration include a range of 1 pg / mL to 10 μg / mL, preferably a range of 10 pg to 1 μg / mL, more preferably a range of 150 pg / mL to 300 pg / mL, and even more preferably a range of 150 pg / mL to 250 pg / mL.

[0040] The content of the iron transporter in the culture medium in step R is not particularly limited as long as it is a culture medium that allows the production of the platelets of the present invention by culturing starter cells or the like in the culture medium used in the present invention having such an iron transporter content. For example, －１５ The concentration range is preferably 100 fM to 10 nM, more preferably 1 pM to 2.8 pM, and even more preferably 1 pM to 2.5 pM.

[0041] Furthermore, when iron-binding transferrin is used in the culture medium in step R, its concentration is not particularly limited and may be within the above-mentioned range of iron ion concentration, for example, 25 μg / mL to less than 400 μg / mL, preferably 50 μg / mL to less than 200 μg / mL. It is said that approximately 1.3 μg of iron ions are bound to 1 mg of iron-binding transferrin.

[0042] The culture medium in step R is not particularly limited as long as it is a culture medium that, when the iron ions and iron transporter of the present invention are added, allows the platelets of the present invention to be produced by culturing start cells or the like in the culture medium. However, a chemically synthesized culture medium is preferred from the viewpoints of ease of preparation and prevention of lot-to-lot variation, and the culture medium preferably contains one or more types of sugar(s), one or more types of inorganic salt(s), one or more types of amino acid(s), one or more types of vitamin(s), and one or more types of other components.

[0043] As described above, the culture medium in step R (culture medium for inducing differentiation into megakaryocytic cells) is a culture medium obtained by adding iron ions and an iron transporter to a basal culture medium for mesenchymal cell culture capable of culturing mesenchymal cells. Specific examples of suitable basal culture medium for mesenchymal cell culture used in step R include commercially available chemically synthesized culture mediums such as Iscove's Modified Dulbecco's Medium (IMDM), RPMI 1640 culture medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Eagle's Medium (BME), and F12 culture medium; a culture medium obtained by mixing two or more of these culture mediums in an appropriate ratio, such as DMEM / F12 culture medium (a culture medium obtained by mixing DMEM and F12 culture medium at a 1:1 ratio); or a culture medium obtained by adding nucleic acids such as nucleotides, penicillin, streptomycin, etc. to any of these culture mediums. Preferred examples include culture media further supplemented with antibiotics and L-glutamine, and more preferred examples include culture media in which antibiotics (preferably penicillin G sodium, streptomycin sulfate, or a penicillin-streptomycin solution) and L-glutamine are further added to IMDM or RPMI1640 culture media. Of these, particularly preferred examples include culture media in which antibiotics (preferably penicillin G sodium, streptomycin sulfate, or a penicillin-streptomycin solution) and L-glutamine are further added to IMDM.

[0044] Particularly suitable basal culture medium for mesenchymal cell culture to be used as the culture medium in step R includes a culture medium in which 2 mM (final concentration) of L-glutamine and 100 U / mL (final concentration) of penicillin-streptomycin solution are added to IMDM having a composition described below (hereinafter referred to as a "particularly suitable basal culture medium to be used as the culture medium in step R"), and a culture medium containing each component independently at a concentration within the range of 70 to 130% by weight (preferably within the range of 80 to 120% by weight) of the concentration of each component in the particularly suitable basal culture medium to be used as the culture medium in step R.

[0045] (Composition of IMDM) 0.4 mM glycine, 0.281 mM L-alanine, 0.398 mM L-arginine hydrochloride, 0.167 mM L-asparagine, 0.226 mM L-aspartic acid, 0.381 mM L-cystine dihydrochloride, 0.51 mM L-glutamic acid, 4 mM L-glutamine, 0.2 mM L-histidine hydrochloride monohydrate, 0.802 mM L-isoleucine, 0.802 mM L-leucine, 0.798 mM L-lysine hydrochloride, 0.201 mM L-methionine, 0.4 mM L-phenylalanine, 0.348 mM L-proline, 0.4 mM L-serine, 0.798 mM L-Threonine, 0.0784 mM, L-Tryptophan, 0.462 mM, L-Tyrosine disodium dihydrate, 0.803 mM, L-Valine, 0.0000533 mM, Biotin, 0.0286 mM, Choline chloride, 0.00839 mM, D-Calcium pantothenate, 0.00907 mM, Folic acid, 0.0328 mM, Nicotinamide, 0.0196 mM, Pyridoxal hydrochloride, 0.00106 mM, Riboflavin, 0.119 mM, Thiamine hydrochloride, 0.0000096 mM, Vitamin B12, 0.04 mM, i-Inositol, 1.49 mM, Calcium chloride anhydrous, 0.84 mM, Magnesium sulfate anhydrous, 4.4 mM, Potassium chloride, 0.000752 mM Potassium nitrate, 36 mM sodium bicarbonate, 77.59 mM sodium chloride, 0.906 mM sodium dihydrogen phosphate monohydrate, 0.0000658 mM sodium selenite pentahydrate, 25 mM D-glucose, 25.03 mM HEPES, 0.0399 mM phenol red, 1 mM sodium pyruvate.

[0046] A particularly suitable culture medium for use in step R can be a culture medium obtained by adding iron ions and an iron transporter to a particularly suitable basal culture medium for use in step R, and more preferably, a culture medium obtained by adding iron-binding transferrin as the sole active ingredient to a particularly suitable basal culture medium for use in step R.

[0047] The culture medium in step R may contain TPO, bovine serum albumin (BSA), LDL cholesterol, insulin, 2-β-mercaptoethanol, etc., but omitting such components provides greater benefits, such as enabling the production of the platelet-like cell population of the present invention at lower cost. That is, MKLI culture medium, a conventional culture medium for inducing differentiation into megakaryocytic cells, contains, in addition to IMDM culture medium, TPO, BSA, LDL cholesterol, insulin, and 2-β-mercaptoethanol (hereinafter, these components are also collectively referred to as the "five components") (Matsubara Y, Murata M, Ikeda Y. Culture of megakaryocytes and platelets from subcutaneous adipose tissue and a preadipocyte cell line. Methods Mol Biol. 2012; 788: 249-258.), but omitting these five components in the culture medium in step R enables the production of the platelet-like cell population of the present invention at lower cost. The present inventors have experimentally confirmed that BSA, LDL cholesterol, insulin, and 2-β-mercaptoethanol are not necessary when inducing differentiation of start cells into megakaryocytes or platelets. Among the culture media in step R (i.e., "culture media for inducing differentiation into megakaryocytic cells containing iron ions and an iron transporter"), a culture medium that does not contain bovine serum albumin, LDL cholesterol, deoxyribonucleotide triphosphate, or 2-mercaptoethanol, but contains human serum albumin, iron-binding transferrin, insulin, and monothioglycerol, is referred to herein as a "modified culture medium for inducing differentiation into megakaryocytic cells." Note that, if the start cells or the like are able to intracellularly incorporate iron ions in the culture medium even in the absence of an iron transporter, the culture medium used in the present invention can also be referred to as a "culture medium for inducing differentiation into megakaryocytic cells containing iron ions" rather than a "culture medium for inducing differentiation into megakaryocytic cells containing iron ions and an iron transporter."

[0048] Particularly preferred embodiments of the culture medium in step R include a culture medium obtained by adding human serum albumin, iron-binding transferrin, insulin, and monothioglycerol to a particularly preferred basal culture medium used for the culture medium in step R, and further include a culture medium that does not contain bovine serum albumin, LDL cholesterol, deoxyribonucleotide triphosphate, or 2-mercaptoethanol.More specific examples include a modified MKLI culture medium having the following composition, and a culture medium containing each component at a concentration that is independently within the range of 70% to 130% (preferably within the range of 80 to 120% by weight) of the concentration of each component in the culture medium. The modified MKLI culture medium can be prepared by adding 2 mM L-glutamine (Life Technologies), 100 U / mL penicillin-streptomycin solution (Life Technologies), 0.5% human serum albumin (Sigma), 200 μg / mL iron-saturated transferrin (Sigma), 10 μg / mL insulin (Sigma), and 20 μM monothioglycerol (Wako) to IMDM culture medium (Iscove's Modified Dulbecco's Medium, Life Technologies). A modified MKLI culture medium with such a composition, or a culture medium containing each component independently at a concentration within the range of 70% to 130% (preferably within the range of 80 to 120% by weight) of the concentration of each component in the culture medium, is preferably included in the "modified culture medium for inducing differentiation into megakaryocytic cells." The modified MKLI culture medium differs from the conventional MKLI culture medium in that it uses human serum albumin instead of BSA, does not use LDL cholesterol, does not use dNTPs, and uses monothioglycerol instead of mercaptoethanol.

[0049] In the above step R, the conditions for culturing the starter cells, etc. in a differentiation-inducing culture medium for megakaryocytic cells containing iron ions and an iron transporter are not particularly limited as long as platelets can be produced by culturing the starter cells, etc. in the culture medium in step R, but the culture temperature can be typically in the range of 12 to 45°C, preferably in the range of 15 to 37°C, and the culture period can be typically in the range of 4 to 25 days, preferably in the range of 5 to 17 days, and can also be in the range of 8 to 17 days.

[0050] The above-described method for producing platelets preferably further comprises a step of increasing the number of cells by maintaining the starter cells, etc., before culturing them in the culture medium in step R. Including such a maintenance culture step makes it possible to increase the number of starter cells, etc., that can be used for culture in the culture medium in step R, as described below, and thus significantly increase the platelet yield of the present invention relative to the number of starter cells, etc., initially prepared. The culture medium used for such maintenance culture is not particularly limited as long as it is a culture medium that can grow the starter cells, etc., and examples thereof include the aforementioned basal culture medium for mesenchymal cell culture (containing no iron ions or iron transporters). When performing maintenance culture, it is preferable to use a basal culture medium for mesenchymal cell culture containing serum or serum components. During the maintenance culture step, it is preferable to appropriately perform passaging and culture medium replacement.

[0051] The method for collecting platelets from the culture in step R is not particularly limited, and examples thereof include a method of separating a culture supernatant containing platelets from the culture, and a method of separating the platelets of the present invention based on molecular size using a filter or the like.

[0052] The above-mentioned method for producing platelets may further include, during or after collection of platelets from the culture, (a) a step of selecting platelets having a specific cell surface marker profile from the platelets (i.e., platelets expressing a specific or specific combination of cell surface markers) using the presence or absence of a specific or specific combination of cell surface markers as an indicator, or (b) a step of increasing or decreasing the proportion of platelets having a specific cell surface marker profile among the platelets, but from the viewpoint of simplicity, etc., it is preferable not to include such a step. The proportion of platelets expressing a specific or specific combination of cell surface markers among platelets that have undergone such a selection step or an increasing or decreasing step includes the proportion of cells positive for the specific cell surface markers listed herein, or all combinations of such proportions.

[0053] There are no particular limitations on (a) the method for selecting platelets having a specific cell surface marker profile from platelets, or (b) the method for increasing or decreasing the proportion of platelets having a specific cell surface marker profile among platelets. However, from the perspective of more simply and quickly selecting platelets having a desired cell surface marker profile or increasing or decreasing the proportion of platelets having a desired cell surface marker profile, a preferred example is a method for selecting platelets having a specific cell surface marker profile using antibodies (preferably labeled antibodies, more preferably fluorescently labeled antibodies) against each of the above-mentioned cell surface markers and using the presence or absence of specific binding of each of these antibodies as an indicator.

[0054] The phrase "selecting using the presence or absence of specific antibody binding as an indicator" means that, for positive cell surface markers in the profile, platelets to which an antibody against the marker specifically binds are selected, and for negative cell surface markers in the profile, platelets to which an antibody against the marker does not specifically bind are selected. Methods for selecting platelets having a specific cell surface marker profile using the presence or absence of specific antibody binding as an indicator are not particularly limited, and include methods using a cell sorter, magnetic beads, or a cell adsorption column, among others. A preferred example is a method using a cell sorter, which is simpler and faster. Methods using a cell sorter are based on flow cytometry and are well known to those skilled in the art. Specific methods are described in the instruction manual for the cell sorter as well as in JP-A-2009-513161. Methods using magnetic beads are well known to those skilled in the art, such as magnetic separation methods. Specific examples include contacting magnetic beads carrying a specific antibody with cells, then recovering the magnetic beads with a magnet to separate platelets that specifically bind to the specific antibody. Furthermore, methods using cell adsorption columns are well known to those skilled in the art, and a specific example of such a method is to contact platelets with a cell adsorption column carrying a specific antibody, and allow platelets other than the target platelets to be adsorbed onto the column.

[0055] Platelets produced by the above-mentioned method for producing platelets (platelets produced from ASCL) co-express one or more types of platelet surface markers and one or more types of mesenchymal cell surface markers.

[0056] In the present invention, "platelets co-expressing one or more types of platelet surface markers and one or more types of mesenchymal cell surface markers" refers to a platelet population containing platelets expressing one or more types of platelet surface markers (platelets that are positive for one or more types of platelet surface markers) and platelets expressing one or more types of mesenchymal cell surface markers (platelets that are positive for one or more types of mesenchymal cell surface markers). The platelet population may contain platelets that are positive for one or more types of platelet surface markers and platelets that are positive for one or more types of mesenchymal cell surface markers, respectively, as separate platelets. However, the platelet population is preferably a platelet population that is positive for one or more types of platelet surface markers and at least partially contains platelets that are positive for one or more types of mesenchymal cell surface markers.

[0057] In the present invention, a platelet population containing "platelets expressing one or more types of platelet surface markers (platelets positive for one or more types of platelet surface markers)" includes a platelet population containing platelets expressing at least CD29, and preferably further includes a platelet population containing platelets expressing one or more types of platelet surface markers (preferably three types) selected from the group consisting of CD49b, CD42b, and CD41. In the present invention, a "platelet population containing platelets expressing two or more types of platelet surface markers" refers to a platelet population containing platelets expressing any one of the two or more types of platelet surface markers (platelets that are positive for any one type of platelet surface marker) and platelets expressing any one or more other types of platelet surface markers (platelets that are positive for any one or more other types of platelet surface markers). The platelet population may contain only platelets that are positive for any one type of platelet surface marker and platelets that are positive for any one or more other types of platelet surface markers, but is preferably a platelet population that contains, at least in part, platelets that are positive for any one type of platelet surface marker and are positive for any one or more other types of platelet surface markers.

[0058] Specific examples of platelet populations containing platelets expressing the platelet surface marker CD29 include platelet populations in which the proportion of CD29-positive platelets is preferably 60% or more, more preferably 70% to 90%, and even more preferably 75% to 85%. Specific examples of platelet populations containing platelets expressing one or more (preferably three) platelet surface markers selected from the group consisting of CD49b, CD42b, and CD41 in addition to CD29 include platelet populations in which the proportion of positive platelets for one or more (preferably three) platelet surface markers selected from the group consisting of CD49b, CD42b, and CD41 satisfies the following numerical ranges: The percentage of CD49b-positive platelets is preferably 30% or more (more preferably 30-85%, even more preferably 30-70%); the percentage of CD42b-positive platelets is preferably 5% or more (more preferably 6-80%, even more preferably 7-60%, even more preferably 8-40%); the percentage of CD41-positive platelets is preferably 20% or more (more preferably 20-85%, even more preferably 30-70%);

[0059] In the present invention, the platelet population containing "platelets expressing one or more types of mesenchymal cell surface markers (platelets positive for one or more types of mesenchymal cell surface markers)" is not particularly limited as long as it is a platelet population containing platelets expressing at least CD90, but preferably includes a platelet population containing platelets further expressing one or more types (preferably three or more, more preferably five or more, even more preferably seven or more, more preferably eight) of mesenchymal cell surface markers selected from the group consisting of CD13, CD26, CD44, CD73, CD77, CD81, CD95, and CD164. In the present invention, a "platelet population containing platelets expressing two or more types of mesenchymal cell surface markers" refers to a platelet population containing platelets expressing any one of the two or more types of mesenchymal cell surface markers (platelets that are positive for any one type of mesenchymal cell surface marker) and platelets expressing any one or more other types of mesenchymal cell surface markers (platelets that are positive for any one or more other types of mesenchymal cell surface markers). The platelet population may contain only platelets that are positive for any one type of mesenchymal cell surface marker and platelets that are positive for any one or more other types of mesenchymal cell surface markers, as separate platelets, but is preferably a platelet population that contains at least a portion of platelets that are positive for any one type of mesenchymal cell surface marker and any one or more other types of mesenchymal cell surface markers.

[0060] Specific examples of platelet populations containing platelets expressing the mesenchymal cell surface marker CD90 include platelet populations in which the percentage of CD90-positive platelets is preferably 30% or more, more preferably 30% to 90%, and even more preferably 35% to 70%. Furthermore, preferred examples include platelet populations in which the percentage of positive platelets for one or more (preferably three or more, more preferably five or more, even more preferably seven or more, and more preferably eight) mesenchymal cell surface markers other than CD90 are selected from the group consisting of CD13, CD26, CD44, CD73, CD77, CD81, CD95, and CD164, satisfying the following numerical ranges: The percentage of CD13-positive platelets is 30% or more (preferably 30-80%, more preferably 35-65%); The percentage of CD26-positive platelets is 15% or more (preferably 15-60%, more preferably 20-45%); The percentage of CD44-positive platelets is 30% or more (preferably 30-80%, more preferably 35-65%); The percentage of CD73-positive platelets is 40% or more (preferably 40-95%, more preferably 45-80%); The percentage of CD95-positive platelets is 20% or more (preferably 20-70%, more preferably 25-55%); The percentage of CD164-positive platelets is 15% or more (preferably 15-55%, more preferably 20-40%);

[0061] As described above, the platelet population produced by the above-mentioned method for producing platelets (preferably a platelet population produced from ASCL) preferably includes a platelet population that is positive for one or more platelet surface markers and that at least partially contains platelets that are positive for one or more mesenchymal cell surface markers, and specifically, a platelet population that satisfies the following percentages of positive platelets: the percentage of platelets that are positive for CD42b and CD90 is 3% or more (preferably 3 to 80%, more preferably 5 to 40%, and even more preferably 5 to 20%);

[0062] A preferred embodiment of the platelet population produced by the above-described platelet production method (preferably, a platelet population produced from ASCL) further includes a platelet population in which the expression of one or more specific platelet surface markers (hereinafter also referred to as "low-expressed platelet surface markers") is reduced compared to the expression in a normal platelet population. A "platelet population in which the expression of one or more low-expressed platelet surface markers is reduced compared to the expression in a normal platelet population" refers to a platelet population in which the proportion of platelets positive for one or more low-expressed platelet surface markers is lower than the proportion in a normal platelet population. Examples of such "low-expressed platelet surface markers" include one or more (preferably, three or more, more preferably five or more) markers selected from the group consisting of CD9, CD36, CD41 / 61, CD61, and CD147. CD41 / 61 refers to a complex of CD41 and CD61, and CD41 / 61-positive platelets refer to platelets expressing the CD41 and CD61 complex.

[0063] A preferred example of a platelet population in which the expression of one or more low-expression platelet surface markers is reduced compared to the expression thereof in a normal platelet population is a platelet population in which the proportion of positive platelets satisfies the following numerical ranges for one or more (preferably three or more, more preferably five or more) low-expression platelet surface markers selected from the group consisting of CD9, CD36, CD41 / 61, CD61, and CD147: The percentage of CD9-positive platelets is 30% or less (preferably 0-20%, more preferably 0.5-15%); The percentage of CD36-positive platelets is 40% or less (preferably 0-30%, more preferably 0.5-20%); The percentage of CD41 / 61-positive platelets is 60% or less (preferably 0-40%, more preferably 0.5-25%); The percentage of CD61-positive platelets is 30% or less (preferably 0-10%, more preferably 0.1-8%); The percentage of CD147-positive platelets is 50% or less (preferably 0-40%, more preferably 1-30%);

[0064] A preferred embodiment of the platelet population produced by the above-described method for producing platelets (preferably, a platelet population produced from ASCL) includes a platelet population that contains platelets positive for one or more platelet surface markers and platelets positive for one or more mesenchymal cell surface markers, and further contains platelets positive for one or two of the activated platelet surface markers CD107a and CD107b. Platelets positive for activated platelet surface markers may be platelets separate from platelets positive for one or more platelet surface markers or platelets positive for one or more mesenchymal cell surface markers. However, the platelet population of the present invention is preferably a platelet population that at least partially contains platelets positive for activated platelet surface markers and one or more platelet surface markers, or platelets positive for activated platelet surface markers and one or more mesenchymal cell surface markers.

[0065] Specific examples of platelet populations containing platelets positive for one or two of the activated platelet surface markers CD107a and CD107b include platelet populations that satisfy the following ranges of the percentage of positive platelets for one or two of the activated platelet surface markers CD107a and CD107b: the percentage of CD107a-positive platelets is 15% or more (more preferably 25-80%, even more preferably 45-70%); the percentage of CD107b-positive platelets is 10% or more (more preferably 15-70%, even more preferably 20-55%);

[0066] The platelet population produced by the above-described method for producing platelets (preferably, a platelet population produced from ASCL) may be (a) a platelet population in which platelets having a specific cell surface marker profile (i.e., platelets expressing a specific or specific combination of cell surface markers) have been selected, or (b) a platelet population in which the proportion of platelets having a specific cell surface marker profile has been increased or decreased, using the presence or absence of a specific or specific combination of cell surface markers as an indicator. In such a platelet population, the proportion of platelets expressing a specific or specific combination of cell surface markers includes the proportion of platelets positive for the specific cell surface markers listed herein, or any combination of such proportions.

[0067] In a preferred embodiment of the platelet population produced by the above-described method for producing platelets (preferably a platelet population produced from ASCL), the amount of TGF-β (transforming growth factor β) produced by the following method is 1×10 platelets or more. ８ Examples of the platelet population include a platelet population having a TGF-β production level of 0.1 pg or more, preferably 1 pg or more, more preferably 2.5 pg or more, even more preferably 5 pg or more, more preferably 7.5 pg or more, even more preferably 10 pg or more, and even more preferably 15 pg or more per platelet. (Method for measuring the amount of TGF-β produced) ８ Platelets were collected in phosphate-buffered saline containing 10 mM CaCl ２ After 15 minutes of incubation, the amount of TGF-β in the phosphate buffered saline solution is measured. The PBS solution used for the suspension contains 8 g / L NaCl, 0.2 g / L KCl, 1.44 g / L NaCl, and ２ HPO ４ , 0.24g / L KH ２ P.O. ４ (pH 7.4) is preferred.

[0068] The platelet-rich plasma of the present invention can be used for any purpose, such as blood transfusion and regenerative medicine.

[0069] The present invention will be specifically described based on the following examples, but the present invention is not limited to these examples.

[0070] Test 1. [Evaluation of platelet function in commercially available platelet preparations] Two bags of commercially available platelet preparations (plasma containing platelets) (manufactured by the Japanese Red Cross Society) were prepared. The shelf life of these platelet preparations was four days. One bag of platelet preparations was stored at room temperature, and the platelet preparation collected in a tube after three days of storage was designated platelet sample A, while the platelet preparation collected in a tube after 14 days of storage was designated platelet sample B. Meanwhile, another bag of platelet preparations was stored at 4°C, and the platelet preparation collected in a tube after 14 days of storage was designated platelet sample C. The platelet concentrations in the plasma of platelet samples A and B were 6.1 x 10 ８ The platelet concentration in the plasma of platelet sample C was 9.9 × 10 ７ The number was 1 / mL.

[0071] Platelet samples B, C, and A were each placed in a tube and centrifuged at 1200 G for 10 minutes at 25°C. After slow deceleration, each supernatant was collected into a separate tube. The supernatant was collected from each tube and designated as Supernatant Sample 1 (supernatant of platelet sample B), Supernatant Sample 2 (supernatant of platelet sample C), and Supernatant Sample 3 (supernatant of platelet sample A) before calcium stimulation (i.e., contact with a solution containing a calcium-containing compound).

[0072] To the platelets remaining in each tube, a volume of 10 mM calcium chloride solution equal to the volume of the solution before centrifugation at 1200 G was added. After addition, each tube was placed in a vortex mixer or Disruptor Jenny (registered trademark) and stirred at 2000 rpm for 10 seconds, then allowed to stand for 15 minutes. Each tube was centrifuged at 1200 G for 10 minutes at 25°C, and after slow deceleration, each supernatant was collected in a separate tube and designated post-calcium-stimulation supernatant samples 4 to 6.

[0073] The VEGF concentrations in supernatant samples 1 to 3 before calcium stimulation and in supernatant samples 4 to 6 after calcium stimulation were measured by ELISA using a commercially available anti-VEGF antibody. The results are shown in Table 1.

[0074]

[0075] The results in Table 1 show that when the storage period was 14 days, which far exceeded the expiration period of 4 days, the VEGF concentration was below the detection limit (supernatant samples 4 and 5), and no VEGF release from platelets was observed, even when platelets were contacted with a solution containing a calcium-containing compound and calcium-stimulated (supernatant samples 4 and 5). On the other hand, when the storage period was 3 days, which was within the expiration period, the VEGF concentration after contacting platelets with a solution containing a calcium-containing compound and calcium-stimulating was 189.98 pg / mL, and VEGF release from platelets was observed (supernatant sample 6). These results demonstrate that the method of the present invention can evaluate platelet function simply and with high accuracy.

[0076] Test 2. [Evaluation of ASCL-PLC Function] (Production of Platelets Derived from an Adipose Tissue-Derived Mesenchymal Stem Cell Line) An adipose tissue-derived mesenchymal stem cell line (ASCL) was prepared according to the method described in the Examples of International Publication WO 2017 / 094260. The obtained ASCL were cultured in modified MKLI medium for 14 days to differentiate the ASCL into platelets. Platelets were collected from the culture medium and suspended in a solvent to prepare a platelet preparation derived from an adipose tissue-derived mesenchymal stem cell line ("ASCL-PLC"). The platelet concentration in this ASCL-PLC preparation was 4.0 x 10 ８ The number was 1 / mL.

[0077] (Evaluation of Platelet Function) Two bags of the ASCL-PLC preparation produced above were prepared. One bag of the ASCL-PLC preparation was stored at 4°C, and a platelet sample collected in a tube after 21 days of storage was designated as platelet sample D before calcium stimulation. Meanwhile, another bag of the ASCL-PLC preparation was stored at -80°C, and a platelet sample collected in a tube after 21 days of storage was designated as platelet sample E before calcium stimulation.

[0078] Each tube contained 200 μL of platelet sample D and E (i.e., 8.0 × 10 platelets). ７The tubes (each containing 1000 pieces of platelets) were centrifuged at 1200 G for 10 minutes at 25°C, and after slow deceleration, the supernatants were collected in separate tubes. The supernatants were collected from each tube and designated as Supernatant Sample 7 (supernatant of platelet sample D) and Supernatant Sample 8 (supernatant of platelet sample E) before calcium stimulation.

[0079] To the platelets remaining in each tube, a volume of 10 mM calcium chloride solution equal to the volume of the solution before centrifugation at 1200 G was added. After addition, each tube was placed in a vortex mixer or Disruptor Jenny (registered trademark) and stirred at 2000 rpm for 10 seconds, then allowed to stand for 15 minutes. Each tube was centrifuged at 1200 G for 10 minutes at 25°C, and after slow deceleration, each supernatant was collected into a separate tube and designated post-calcium-stimulation supernatant samples 9 and 10.

[0080] The VEGF concentrations in supernatant samples 7 and 8 before calcium stimulation and in supernatant samples 9 and 10 after calcium stimulation were measured by ELISA using the same method as in Test 1. The results are shown in Table 2.

[0081]

[0082] The results in Table 2 show that even when ASCL-PLC was used as platelets, calcium stimulation by contact with a solution containing a calcium-containing compound detected sufficient concentrations of VEGF, confirming the release of VEGF from the ASCL-PLC (supernatant samples 9 and 10). This demonstrates that the method of the present invention can easily and accurately evaluate platelet function, even when ASCL-PLC is used as platelets. Furthermore, ASCL-PLC released sufficiently high concentrations of VEGF upon calcium stimulation, even after long-term storage of 21 days, regardless of whether the storage temperature was 4°C or -80°C, demonstrating that the excellent platelet function can be maintained for a long period of time.

[0083] Test 3. [Evaluation of Peripheral Blood Platelet Function] (Preparation of Peripheral Blood Platelets) 3 mL of an acid citrate dextrose solution (ACD-A solution) as an anticoagulant was dispensed into two 20 mL syringes. Approximately 20 mL of blood was collected from each of two healthy volunteers using the syringes described above, and each whole blood sample was dispensed into a polypropylene tube. The two polypropylene tubes containing each whole blood sample were placed in a centrifuge and centrifuged at 200 G for 10 minutes. After slow deceleration, the supernatant platelet-rich plasma (PRP) (approximately 3 mL: a volume ratio of 1:0.4 based on the total volume of the liquid after centrifugation) was collected into each tube, preparing two types of peripheral blood platelets (platelet samples F and G). The platelet concentration of platelet sample F was 1.2 x 10 ８ The platelet concentration of platelet sample G was 7.4 × 10 ７ The number was 1 / mL.

[0084] Each tube containing 3000 μL of platelet sample F and 3000 μL of platelet sample G was centrifuged at 1200 G for 10 minutes at 25°C. After slow deceleration, the supernatants were collected in separate tubes. The supernatants were collected from each tube and designated pre-calcium-stimulation supernatant sample 11 (supernatant of platelet sample F) and supernatant sample 12 (supernatant of platelet sample G).

[0085] To the platelets remaining in each tube, a volume of 10 mM calcium chloride solution equal to the volume of the solution before centrifugation at 1200 G was added. After addition, each tube was placed in a vortex mixer or Disruptor Jenny (registered trademark) and stirred at 2000 rpm for 10 seconds, then allowed to stand for 15 minutes. Each tube was centrifuged at 1200 G for 10 minutes at 25°C, and after slow deceleration, each supernatant was collected in a separate tube and designated post-calcium-stimulation supernatant samples 13 and 14.

[0086] The VEGF concentrations in Samples 11 and 12 before calcium stimulation and in Samples 13 and 14 after calcium stimulation were measured by ELISA using the same method as in Test 1. The results are shown in Table 3.

[0087]

[0088] The results in Table 3 demonstrate that even when peripheral blood platelets are used as platelets, the method of the present invention makes it possible to evaluate platelet function simply and with high accuracy.

[0089] According to the present invention, a method for evaluating platelet function simply and with high accuracy can be provided.

Claims

1. A method for evaluating platelet function, comprising: step A: contacting platelets with a solution containing a calcium-containing compound; and step B: detecting or quantifying cytokines released by the platelets after step A.

2. The method according to claim 1 or 2, wherein the calcium concentration of the solution containing the calcium-containing compound is 0.01 to 100 mM.

3. The method according to claim 1 or 2, wherein the platelets are contacted with the solution containing the calcium-containing compound for a period of 0.1 minutes to 3 hours.

4. The method of claim 1 or 2, wherein the cytokine is one or more selected from the group consisting of VEGF (Vascular Endothelial Growth Factor), FGF (Fibroblast Growth Factor), TGF (Transforming Growth Factor), EGF (Epidermal Growth Factor), PDGF (Platelet-Derived Growth Factor), IGF (Insulin-like Growth Factor), and HGF (Hepatocyte Growth Factor).

5. The method according to claim 1 or 2, further comprising, after step B, step C, of ​​assessing that the platelets have platelet function when the platelets release cytokines upon contact with a solution containing a calcium-containing compound.

6. The method according to claim 1 or 2, further comprising, before step A, a step P of centrifuging the platelet-containing liquid to isolate the platelets.

7. The method according to claim 1 or 2, further comprising a step Q after step A and before step B, in which the platelet-containing liquid is centrifuged to isolate the platelets.

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