Preservation solution for mammalian cells

Abstract

To provide a new preservation solution that has better preservation stability than conventional preservation solutions and can preserve mammalian cells for a long period of time.SOLUTION: A method for preserving mammalian cells comprises a step of preserving mammalian cells, such as mesenchymal stem cells, at 0 to 40°C, for 1 to 28 days in liquid that contains an ascorbic acid or a derivative thereof or a salt thereof and trehalose, and optionally dextran in an isotonic solution, such as a lactated Ringer's solution.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to the preservation of platelets using a preservation solution containing niacin or its salt and ascorbic acid or its salt.

Background Art

[0002] Platelet preparations are administered to patients with decreased platelets, in addition to bleeding during surgery or trauma. Currently, platelet preparations are manufactured from blood obtained by blood donation, but there is concern that the amount of blood donation may decrease due to changes in the population composition, resulting in a shortage of platelet preparations.

[0003] In addition, when the blood donor has an infectious disease such as bacteria, there is a possibility that the blood is contaminated with bacteria, so there is a risk of infectious diseases due to the administration of platelet preparations contaminated with bacteria. For this reason, methods for manufacturing platelets in vitro have been developed (Non-Patent Document 1), and various technologies for stable mass production have been established (Patent Documents 1 and 2).

[0004] Platelet preparations are manufactured by mixing platelets with an appropriate preservation solution and filling them into blood bags or the like. Since the morphology and function of platelets change rapidly at low temperatures, platelet preparations are stored with shaking at room temperature (20 to 24°C) and used. However, it is known that various problems such as a decrease in platelet function occur during storage, and long-term storage has been impossible. Therefore, there is a demand for the development of a preservation solution that prevents a decrease in platelet function and enables an extension of the storage period of platelet preparations.

[0005] Mesenchymal stem cells are somatic stem cells present in bone marrow, adipose tissue, etc., and have the ability to differentiate into bone, cartilage, fat, etc. Therefore, mesenchymal stem cells have attracted attention as a promising cell source in transplantation therapy, and the development of culture and preservation methods has been underway (Patent Documents 3 and 4). More recently, various cancer immunotherapies using T cells have been developed and clinical trials have been conducted (Non-Patent Document 2). However, it has been reported that when T cells are cryopreserved, the expression of PD-1 (an immune checkpoint molecule) on the T cell membrane is significantly reduced (Non-Patent Document 3), and the development of a non-cryopreservation method for T cells is required.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0008] An object of the present invention is to provide a novel preservation solution and the like that are superior in preservation properties to conventional preservation solutions and can preserve platelets for a long period of time.

Means for Solving the Problems

[0009] In order to solve the above problems, the present inventors have conducted intensive studies and found that: (1) When platelets are stored with shaking in an isotonic solution containing ascorbic acid (hereinafter sometimes referred to as "vitamin C" or "VC"), deterioration of platelets is suppressed more than in conventional platelet preservation solutions; (2) When niacin (hereinafter sometimes referred to as "vitamin B3" or "VB3") is added to the isotonic solution in combination with VC, deterioration of platelets is suppressed more efficiently; (3) An isotonic solution containing VC and VB3 is also effective for non-freezing preservation of other mammalian cells such as mesenchymal stem cells, megakaryocytes, and T cells; (4) The cell preservation effect by VC and VB3 as described above is inhibited by riboflavin (vitamin B2, hereinafter sometimes referred to as "VB2"), and thus the present invention has been completed.

[0010] That is, the present invention is specified as follows. [1] A mixed solution containing a preservation solution and platelets, the preservation solution containing 10 to 5000 mg / L of niacin or a salt thereof, and 10 to 8000 mg / L of ascorbic acid or a salt thereof, having an osmotic pressure isotonic with the platelets, the mixed solution comprising the preservation solution and the platelets stored in the preservation solution, wherein when the platelets are filled in a blood storage bag together with the preservation solution at a platelet concentration of 1.0×10 9 plts / mL and horizontally shaken at 50 rpm for 10 days at 22°C under light shielding, the preservation solution remains neutral. [2] Platelets preserved by a preservation solution having the following characteristics i) and ii): i) containing 10 to 5000 mg / L of niacin or a salt thereof, and 10 to 8000 mg / L of ascorbic acid or a salt thereof, and having an osmotic pressure isotonic with the platelets; ii) the platelets are filled in a blood storage bag at a concentration of 1.0×10 9When stored with a platelet concentration of plts / mL under light shielding at 22°C with horizontal shaking at 50 rpm for 10 days, the storage solution remains neutral for the platelets. [3] A platelet preparation stored with a storage solution having the following characteristics (i) and (ii): (i) containing 10 to 5000 mg / L of niacin or a salt thereof and 10 to 8000 mg / L of ascorbic acid or a salt thereof, and having an osmotic pressure isotonic with the platelets; (ii) when the platelets are contained in a blood storage bag at a concentration of 1.0×10 9 When stored with a platelet concentration of plts / mL under light shielding at 22°C with horizontal shaking at 50 rpm for 10 days, the storage solution remains neutral for the platelet preparation. [4] A storage solution for storing platelets, containing 10 to 5000 mg / L of niacin or a salt thereof and 10 to 8000 mg / L of ascorbic acid or a salt thereof, having an osmotic pressure isotonic with the platelets, and when the platelets are filled in a blood storage bag together with the storage solution at a concentration of 1.0×10 9 When stored with a platelet concentration of plts / mL under light shielding at 22°C with horizontal shaking at 50 rpm for 10 days, the storage solution remains neutral for the storage solution. [5] A mixed solution containing a storage solution and platelets or megakaryocytes, containing 10 to 5000 mg / L of niacin or a salt thereof and 10 to 8000 mg / L of ascorbic acid or a salt thereof in an isotonic solution, and not containing vitamin B2 or a salt thereof, for storing platelets or megakaryocytes at 20 to 24°C for 1 to 15 days, the mixed solution containing the storage solution and the platelets or megakaryocytes stored in the storage solution. [6] Platelets stored with a storage solution, the storage solution being a storage solution for storing platelets at 20 to 24°C for 1 to 15 days, not containing 10 to 5000 mg / L of niacin or a salt thereof, 10 to 8000 mg / L of ascorbic acid or a salt thereof, and vitamin B2 or a salt thereof. [7]A platelet preparation preserved with a preservation solution, wherein the preservation solution contains 10 to 5000 mg / L of niacin or a salt thereof and 10 to 8000 mg / L of ascorbic acid or a salt thereof in an isotonic solution, does not contain vitamin B2 or a salt thereof, and is a preservation solution for preserving platelets at 20 to 24°C for 1 to 15 days, said platelet preparation. [8]A method for preserving platelets, comprising the step of preserving platelets in a preservation solution containing 10 to 5000 mg / L of niacin or a salt thereof and 10 to 8000 mg / L of ascorbic acid or a salt thereof, and having an osmotic pressure isotonic with the platelets, wherein the preservation solution is such that when the platelets are filled in a blood storage bag together with the preservation solution at a platelet concentration of 1.0×10 9 plts / mL and horizontally shaken at 50 rpm for 10 days at 22°C under light shielding, the preservation solution remains neutral, said method for preserving platelets. Also, as another aspect of the present invention, there can be mentioned i) an isotonic solution containing ascorbic acid or a derivative thereof or a salt thereof and trehalose for use in preserving mammalian cells, and ii) the use of an isotonic solution containing ascorbic acid or a derivative thereof or a salt thereof and trehalose for preparing a preservation solution for mammalian cells. Although not the present invention, aspects of related inventions are listed below.

[0011] The related inventions are as follows: (1) a preservation solution for mammalian cells containing niacin or its derivative or their salts and an antioxidant; (2) the preservation solution according to (1) above, wherein the antioxidant is ascorbic acid or its derivative or their salts; (3) the preservation solution according to (1) or (2) above, wherein the concentration of niacin or its derivative or their salts is 1 to 10,000 mg / L; (4) the preservation solution according to (1) or (2) above, wherein the concentration of niacin or its derivative or their salts is 30 to 3,000 mg / L; (5) the preservation solution according to (1) or (2) above, wherein the concentration of niacin or its derivative or their salts is 120 to 1,200 mg / L; (6) the preservation solution according to any one of (2) to (5) above, wherein the concentration of ascorbic acid or its derivative or their salts is 1 to 10,000 mg / L; (7) the preservation solution according to any one of (2) to (5) above, wherein the concentration of ascorbic acid or its derivative or their salts is 30 to 6,000 mg / L; (8) the preservation solution according to any one of (2) to (5) above, wherein the concentration of ascorbic acid or its derivative or their salts is 300 to 3,000 mg / L; (9) the preservation solution according to any one of (1) to (8) above for preserving mammalian cells at 0 to 40°C; (10) the preservation solution according to any one of (1) to (9) above that does not contain vitamin B2 or its derivative or their salts; (11) the preservation solution according to any one of (1) to (10) above, wherein the mammalian cells are platelets or megakaryocytes; (12) the preservation solution according to (11) above, wherein the platelets are purified platelets obtained by a method including the following (A) and (B): (A) a concentration step of concentrating a culture of megakaryocytes; (B) a centrifugation step of centrifuging platelets from the obtained concentrate; (13) the preservation solution according to (11) or (12) above, further containing albumin; (14) the preservation solution according to (13) above, wherein the concentration of albumin is 1.25 to 10% (w / v); (15) the preservation solution according to (13) or (14) above, further containing sugar; (16) the preservation solution according to (15) above, wherein the sugar is glucose; (17) relating to the preservation solution according to any one of (11) to (16) above for preserving platelets or megakaryocytes for 5 to 10 days.

[0012] Furthermore, the related inventions include: (18) the preservation solution according to any one of (1) to (10) above, wherein the mammalian cells are stem cells or immune cells; (19) the preservation solution according to (18) above, wherein the stem cells are mesenchymal stem cells; (20) the preservation solution according to (18) above, wherein the immune cells are T cells; (21) the preservation solution according to any one of (18) to (20) above, which contains niacin or its derivative or their salts and an antioxidant in an isotonic solution; (22) the preservation solution according to (21) above, wherein the isotonic solution is Ringer's lactate solution; (23) the preservation solution according to (21) or (22) above, which further contains trehalose; (24) the preservation solution according to any one of (21) to (23) above, which further contains dextran; (25) the preservation solution according to any one of (18) to (24) above, for preserving stem cells for 1 to 63 days; (26) a powder preparation for preparing the preservation solution according to any one of (1) to (25) above, which contains niacin or its derivative or their salts and an antioxidant.

[0013] Also, the related invention includes a method for preserving mammalian cells, which comprises a step of preserving mammalian cells in a liquid containing (27) niacin or its derivative or their salt and an antioxidant, a method according to (27) above, wherein (28) the antioxidant is ascorbic acid or its derivative or their salt, a method according to (27) or (28) above, wherein (29) the concentration of niacin or its derivative or their salt is 1 to 10,000 mg / L, a method according to (27) or (28) above, wherein (30) the concentration of niacin or its derivative or their salt is 30 to 3,000 mg / L, a method according to (27) or (28) above, wherein (31) the concentration of niacin or its derivative or their salt is 120 to 1,200 mg / L, a method according to any one of (28) to (31) above, wherein (32) the concentration of ascorbic acid or its derivative or their salt is 1 to 10,000 mg / L, a method according to any one of (28) to (31) above, wherein (33) the concentration of ascorbic acid or its derivative or their salt is 30 to 6,000 mg / L, a method according to any one of (28) to (31) above, wherein (34) the concentration of ascorbic acid or its derivative or their salt is 300 to 3,000 mg / L, a method according to any one of (27) to (34) above, which is characterized in that (35) mammalian cells are preserved at 0 to 40°C, and a method according to any one of (27) to (35) above, which is characterized in that (36) the liquid does not contain vitamin B2 or its derivative or their salt.

[0014] Furthermore, related inventions include: (37) the method according to any one of (27) to (36) above, wherein the mammalian cells are platelets or megakaryocytes; (38) the method according to (37) above, wherein the platelets are purified platelets obtained by a method comprising the following (A) and (B): (A) a concentration step of concentrating a culture of megakaryocytes; (B) a centrifugation step of centrifuging platelets from the obtained concentrate; (39) the method according to (37) or (38) above, wherein the solution further contains albumin; (40) the method according to (39) above, wherein the concentration of albumin is 1.25 to 10% (w / v); (41) the method according to (39) or (40) above, wherein the solution further contains sugar; (42) the method according to (41) above, wherein the sugar is glucose; (43) the method according to any one of (37) to (42) above, characterized in that the platelets or megakaryocytes are stored for 5 to 10 days; (44) the method according to any one of (27) to (36) above, wherein the mammalian cells are stem cells or immune cells; (45) the method according to (44) above, wherein the stem cells are mesenchymal stem cells; (46) the method according to (44) above, wherein the immune cells are T cells; (47) the method according to any one of (44) to (46) above, wherein the solution is an isotonic solution; (48) the method according to (47) above, wherein the isotonic solution is lactated Ringer's solution; (49) the method according to (47) or (48) above, wherein the solution further contains trehalose; (50) the method according to any one of (47) to (49) above, wherein the solution further contains dextran; (51) the method according to any one of (44) to (50) above, characterized in that the stem cells are stored for 1 to 63 days.

Advantages of the Invention

[0015] According to the present invention, compared with conventional preservation solutions, it is possible to efficiently suppress a decrease in the function and viability of mammalian cells during storage. Therefore, according to the present invention, it becomes possible to store mammalian cells such as platelets, which are difficult to cryopreserve, for a long period of time, and to provide a high-quality cell-containing solution for transplantation in medicine.

Brief Description of the Drawings

[0016]

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[0017] The mammalian cell preservation solution of the present invention (hereinafter sometimes referred to as "the preservation solution of the present invention") is not particularly limited as long as it contains niacins and / or antioxidants and is limited to the use of "preserving mammalian cells". Furthermore, the powder preparation of the present invention is not particularly limited as long as it is used to prepare the preservation solution of the present invention, and the method for preserving mammalian cells of the present invention is not particularly limited as long as it includes a step of preserving mammalian cells in the preservation solution of the present invention. In other words, the preservation solution of the present invention is a solution that, when mixed with mammalian cells, The term "preservation solution" refers to a solution that exerts the above-mentioned preservation effect in the case where the preservation solution of the present invention is used, and includes not only a preservation solution that does not yet contain cells to be preserved, but also a preservation solution that already contains cells to be preserved. The preservation solution of the present invention may contain niacins alone or antioxidants alone as an active ingredient for preserving mammalian cells, but is preferably a solution that contains a combination of niacins and antioxidants. Hereinafter, niacins and / or antioxidants may be referred to as "essential protective ingredients of the present invention". Furthermore, additives for preparing the preservation solution or powder formulation of the present invention are also included in the present invention. The additives of the present invention necessarily contain the essential protective ingredients of the present invention. The additives of the present invention do not need to contain the "optional active ingredients" described below.

[0018] The niacin (VB3) in the above-mentioned "niacins" means nicotinic acid and / or nicotinamide. That is, the preservation solution of the present invention may be any of a preservation solution containing nicotinic acid, a preservation solution containing nicotinamide, and a preservation solution containing nicotinic acid and nicotinamide, but a preservation solution containing nicotinic acid is preferred. In addition, niacin (VB3) can be produced by known methods such as chemical synthesis, or commercially available products can also be used. For example, commercially available products such as nicotinic acid injection preparations (manufactured by Toa Eiyo Co., Ltd.) and nicotinamide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) can be mentioned.

[0019] The niacin derivatives in the above-mentioned "niacins" are not particularly limited. For example, tocopherol nicotinate, niceritrol, nicomol, inositol hexanicotinate, 2-chloronicotinamide, 6-methylnicotinamide, 6-aminonicotinamide, N-methylnicotinamide, N,N-dimethylnicotinamide, N-(hydroxymethyl)nicotinamide, quinolinic acid imide, nicotinanilide, N-benzylnicotinamide, N-ethylnicotinamide, phenazone, nicotinaldehyde, isonicotinic acid, methyl isonicotinic acid, thionicotinamide, nialamide, 2-mercaptonicotinic acid, niaprazine, methyl nicotinate, sodium nicotinate, etc. can be preferably mentioned. Furthermore, the "salt of niacin or its derivative" in the above-mentioned "niacins" is not particularly limited, but alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, ammonium salts, organic base salts such as trialkylamine salts, mineral acid salts such as hydrochloride and sulfate, organic acid salts such as acetate, etc. can be preferably mentioned.

[0020] In addition, the above-mentioned "antioxidant" is not particularly limited. For example, ascorbic acid (VC), superoxide dismutase 1, superoxide dismutase 2, superoxide dismutase 3, glutathione, lipoic acid, epigallocatechin gallate, curcumin, metranin, hydroxytyrosol, ubiquinone, catalase, vitamin E, uric acid, etc., their derivatives, or their salts can be preferably mentioned. Among them, ascorbic acid (VC) or its derivatives or their salts (hereinafter may be collectively referred to as "ascorbic acids") can be preferably mentioned, and ascorbic acid (VC) can be particularly preferably mentioned. Ascorbic acid (VC) can be produced by known methods such as chemical synthesis, but commercially available products can also be used. For example, commercially available products such as ascorbic acid injection (manufactured by Sawai Pharmaceutical Co., Ltd.) and L(+)-ascorbic acid standard product (manufactured by FUJIFILM Wako Pure Chemical Corporation) can be mentioned.

[0021] The derivatives of ascorbic acid (VC) in the above-mentioned "ascorbic acids" are not particularly limited. For example, ascorbic acid 2-phosphate, ascorbic acid 2-sulfate, ascorbyl-2-glucoside, ascorbyl-6-glucoside, etc. can be preferably mentioned. Furthermore, the salts of ascorbic acid (VC) or its derivatives in the above-mentioned "ascorbic acids" are not particularly limited, but alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, ammonium salt, organic base salts such as trialkylamine salt, mineral acid salts such as hydrochloride and sulfate, organic acid salts such as acetate, etc. can be preferably mentioned.

[0022] The concentration of niacins contained in the preservation solution of the present invention may be, for example, in the range of 1 to 10000 mg / L in terms of nicotinic acid conversion. Specifically, 10 to 5000 mg / L, 20 to 8000 mg / L, 30 to 6000 mg / L, 30 to 4000 mg / L, 30 to 3000 mg / L, 30 to 2000 mg / L, 30 to 1500 mg / L, 30 to 1200 mg / L can be cited as examples. Among them, it is preferably in the range of 120 to 1200 mg / L.

[0023] The concentration of ascorbic acids contained in the preservation solution of the present invention may be, for example, in the range of 1 to 10,000 mg / L in terms of ascorbic acid, for example, 10 to 8,000 mg / L, 20 to 7,000 mg / L, 30 to 6,000 mg / L, 30 to 5,000 mg / L, 30 to 4,000 mg / L, 30 to 3,000 mg / L, 50 to 3,000 mg / L, 100 to 3,000 mg / L. Among them, it is preferably in the range of 300 to 3,000 mg / L.

[0024] In addition, the preservation solution of the present invention may contain the essential protective component of the present invention in an isotonic solution. The above "isotonic solution" is not particularly limited as long as it is an isotonic solution in which the salt concentration or sugar concentration is adjusted by sodium ions, potassium ions, calcium ions, etc. so as to be almost the same as the osmotic pressure of body fluid or cell fluid. Specifically, physiological saline, physiological saline with a buffering effect (Phosphate buffered saline [PBS], Tris Buffered Saline [TBS], HEPES buffered saline, etc.), Ringer's solution, lactate Ringer's solution inger's solution, acetic acid Ringer's solution, bicarbonate Ringer's solution, etc. can be mentioned. Among them, bicarbonate Ringer's solution or lactate Ringer's solution is preferably used. The isotonic solution can be manufactured based on a known composition, but commercially available products can also be used. Examples of commercially available products include, for example, Otsuka Fresh Food Injection (physiological saline solution, manufactured by Otsuka Pharmaceutical Factory Co., Ltd.), Ringer's Solution "Otsuka" (Ringer's solution, manufactured by Otsuka Pharmaceutical Factory Co., Ltd.), Lactec (registered trademark) Injection (lactate Ringer's solution, manufactured by Otsuka Pharmaceutical Factory Co., Ltd.), Vene (registered trademark) F Infusion Solution (acetic acid Ringer's solution, manufactured by Fuso Pharmaceutical Industries, Ltd.), Vicaneite (registered trademark) Infusion Solution (bicarbonate Ringer's solution, manufactured by Otsuka Pharmaceutical Factory Co., Ltd.), etc. In this specification, "isotonic" means that the osmotic pressure is within the range of 250 to 380 mOsm / L.

[0025] Furthermore, the preservation solution of the present invention may contain albumin or sugar as any active ingredient for the preservation of mammalian cells. As used herein, the term "any active ingredient" means an ingredient that may or may not be included. Albumin and / or sugar may sometimes be referred to as "optional protective ingredients of the present invention". Examples of the above-mentioned "albumin" include human serum albumin (HSA), bovine serum albumin (BSA), fetal bovine serum albumin (FBS), etc., and HSA is preferably used. When the preservation solution of the present invention contains albumin, the concentration of albumin may be within the range of 0.1 to 30 (w / v)%, for example, 1.0 to 20 (w / v)%, 1.0 to 15 (w / v)%, 1.0 to 10 (w / v)%, 1.25 to 10 (w / v)%, 1.25 to 7.25 (w / v)%, 1.5 to 5.0 (w / v)%, 1.5 to 2.5 (w / v)%, 2.0 to 2.5 (w / v)%.

[0026] Examples of the above-mentioned "sugar" include glucose, trehalose, dextran, hydroxyethyl starch, etc. When the preservation solution of the present invention contains glucose, the concentration of glucose may be within the range of 1 to 10000 mg / L, for example, 10 to 8000 mg / L, 20 to 6000 mg / L, 30 to 6000 mg / L, 40 to 6000 mg / L, 50 to 6000 mg / L, 100 to 6000 mg / L, 200 to 6000 mg / L, 500 to 6000 mg / L, 1000 to 6000 mg / L, 2000 to 6000 mg / L, 2000 to 5000 mg / L. When the preservation solution of the present invention contains trehalose, the concentration of trehalose may be 0.1 to 100 g / L, 5 to 80 g / L, 20 to 60 g / L. When the preservation solution of the present invention contains dextran, the concentration of dextran may be 0.1 to 100 g / L, 5 to 80 g / L, 40 to 70 g / L. When the preservation solution of the present invention contains hydroxyethyl starch, the concentration of hydroxyethyl starch may be 1 to 500 g / L, 10 to 100 g / L.

[0027] The preservation solution of the present invention preferably does not contain (a) vitamin B2 (also referred to as VB2 or riboflavin), its derivatives, or their salts (hereinafter sometimes referred to as "vitamin B2 compounds"), (b) a medium or its essential components, or (c) a cell differentiation promoter. Examples of the derivatives of vitamin B2 in the "vitamin B2 compounds" of (a) above include flavin mononucleotide, flavin adenine dinucleotide, riboflavin tetrabutyrate, riboflavin butyrate, riboflavin phosphate (riboflavin phosphate ester), etc. Examples of the salts of vitamin B2 or its derivatives include sodium salts, etc.

[0028] The "culture medium" in (b) above means a cell culture solution that provides the nutrients necessary for the maintenance and growth of cells in an in vitro environment. Examples thereof include Eagle's Minimum Essential (EME) medium, Iscove's Modified Dulbecco's Medium (IMDM), Dulbecco's Modified Eagle Medium (DMEM), TC199 medium, alpha-Minimum Essential Medium (α-MEM), RPMI1640, Ham-F-12, E199, MCDB, Leibovitz L-15, William's E medium, and the like. Further, the "essential components of the culture medium" in (b) above means aqueous nutrients and electrolytes, glycosaminoglycans, swelling agents, energy sources, buffers, antioxidants, membrane stabilizers, antibiotics (or antifungal agents), ATP precursors, cell nutrient supplements, and / or pH indicators. For example, as the "aqueous nutrients and electrolytes", one or more media selected from the above culture media; as the "glycosaminoglycans", chondroitin sulfate, dermatan sulfate, dermatin sulfate, heparin sulfate, heparan sulfate, keratin sulfate, keratan sulfate, or hyaluronic acid; as the "swelling agents", dextran, dextran sulfate, polyvinylpyrrolidone, polyethylene glycol, polyvinyl acetate, hydroxypropylmethylcellulose, or carboxypropylmethylcellulose; as the "energy sources", pyruvate, sucrose, fructose, or dextrose; as the "buffers", bicarbonate buffer or HEPES buffer; as the "antioxidants", 2-mercaptoethanol, glutathione, or alpha-tocopherol; as the "membrane stabilizers", vitamin A, retinoic acid, ethanolamine, phosphoethanolamine, selenium, or transferrin; as the "antibiotics and / or antifungal agents", amphotericin-B, gentamicin sulfate, kanamycin sulfate, neomycin sulfate, nystatin, penicillin, tobramycin, or streptomycin; as the "ATP precursors", adenosine, inosine, or adenine; as the "cell nutrient supplements", cholesterol, L-hydroxyproline, d-biotin, calciferol, niacin, p-aminobenzoic acid, pyridoxine hydrochloride, vitamin B12, Fe(NO 3 ) 3Alternatively, an essential amino acid or a non-essential amino acid, and as the above-mentioned "pH indicator", phenol red can be respectively mentioned.

[0029] The "cell differentiation promoter" in the above (c) means a drug added to a medium or the like in order to obtain cells of a desired type from cells having differentiation ability. For example, retinol, vitamin D2, vitamin D3, vitamin K, retinoic acid, zinc, zinc compounds, calcium, calcium compounds, hydrocortisone, dexamethasone, L-glutamine, ethylene glycol tetraacetic acid (EGTA), proline, non-essential amino acids (NEAA), β-mercaptoethanol, dibutyryl cyclic adenosine monophosphate (db-cAMP), monothioglycerol (MTG), putrescine, dimethyl sulfoxide (DMSO), hypoxanthine, adenine, forskolin, cilostamide, 3-isobutyl-1-methylxanthine, 5-azacytidine, pyruvate, okadaic acid, linoleic acid, ethylenediaminetetraacetic acid (EDTA), anticoagulant citrate dextrose formulation A (ACDA), disodium EDTA, sodium butyrate, glycerophosphate, G418, gentamicin, pentoxifylline (1-(5-oxohexyl)-3,7-dimethylxanthine), indomethacin, tissue plasminogen activator (TPA), etc. can be mentioned. As described above, in the present invention, niacins and antioxidants (including ascorbic acids) are used for the preservation of mammalian cells and are not used as differentiation promoters.

[0030] The "mammalian cells" targeted by the preservation solution of the present invention are not particularly limited as long as they are living cells derived from mammals, and may be primary cells obtained from an organism, or cells obtained by culturing primary cells for one or more generations for proliferation. Specifically, platelets used for the treatment of diseases, injuries, etc., and megakaryocytes used for their production, as well as stem cells, immune cells, etc. used for regenerative medicine, immunotherapy, etc. can be preferably exemplified. Further, examples of the above "mammals" include rodents such as mice, rats, hamsters, guinea pigs, rabbits of the order Lagomorpha, ungulates such as pigs, cows, goats, horses, sheep, carnivores such as dogs, cats, and primates such as humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, chimpanzees, etc. Among them, humans can be preferably exemplified.

[0031] The above "platelets" refer to one of the cell components in blood and mean cell components that are positive for CD41a and CD42b. The platelets targeted by the preservation solution of the present invention may be concentrated platelets obtained by removing red blood cells and white blood cells from mammalian blood (whole blood), or purified platelets artificially produced from cultured megakaryocytes in vitro, but purified platelets are preferred. The method for producing the above purified platelets is not particularly limited, but a method including step (A): a concentration step of concentrating a culture of megakaryocytes; and step (B): a centrifugation step of centrifuging platelets from the obtained concentrate can be preferably exemplified. Further, the "megakaryocytes" also used in such a platelet production method can be induced from pluripotent cells such as induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), nuclear transfer ES cells (ntES cells), embryonic germ cells (EG cells), somatic stem cells, and embryonal carcinoma cells, and can also be induced from hematopoietic stem cells, hematopoietic progenitor cells, CD34-positive cells, megakaryocyte-erythroblast progenitor cells, megakaryocyte progenitor cells, etc. isolated from bone marrow, umbilical cord blood, peripheral blood, etc.

[0032] Further, the above "stem cells" refer to immature cells having self-renewal ability and differentiation / proliferation ability. Stem cells include, according to their differentiation ability, pluripotent stem cells, multipotent Subpopulations such as multipotent stem cells and unipotent stem cells are included. Multipotent stem cells refer to cells that, although they cannot become an individual on their own, have the ability to differentiate into all tissues and cells that make up a living body. Pluripotent stem cells refer to cells that, although not all types, have the ability to differentiate into multiple types of tissues and cells. Unipotent stem cells refer to cells that have the ability to differentiate into specific tissues or cells. The stem cells to be preserved in the preservation solution of the present invention may be any of multipotent stem cells, pluripotent stem cells, and unipotent stem cells. For example, multipotent stem cells such as iPS cells, ES cells, ntES cells, and EG cells, and somatic stem cells such as mesenchymal stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells, and germ stem cells can be mentioned. Among them, mesenchymal stem cells are preferably used. Mesenchymal stem cells can be collected from the bone marrow, adipose tissue, peripheral blood, umbilical cord blood, etc. of mammals by known general methods. In addition, human mesenchymal stem cells can be isolated by culturing and subculturing hematopoietic stem cells, etc. after bone marrow puncture. Among them, human bone marrow-derived mesenchymal stem cells and young pig bone marrow-derived mesenchymal stem cells can be preferably exemplified.

[0033] Furthermore, the above-mentioned "immune cells" refer to cells present in blood and lymph fluid that are involved in the immune system. Examples of the immune cells to be preserved in the preservation solution of the present invention include, for example, T cells, macrophages, dendritic cells, B cells, NK cells, neutrophils, eosinophils, myeloid-derived suppressor cells (MDSC), etc. Among them, T cells are preferably used. "T cells" refer to cells that express an antigen receptor called a T cell receptor (TCR) on their surface. For example, cytotoxic T cells that are CD8-positive cells, helper / regulatory T cells that are CD4-positive cells, naive T cells (CD45RA+CD62L+ cells), central memory T cells (CD45RA-CD62L+ cells), effector memory T cells (CD45RA-CD62L- cells), and terminal effector T cells (CD45RA+CD62L- cells) can be mentioned. T cells can be collected from the peripheral blood, lymph nodes, bone marrow, thymus, spleen, umbilical cord blood, etc. of mammals by known general methods. In addition, commercially available products can also be used.

[0034] The preservation solution of the present invention can be used to preserve mammals in a non-frozen state. When preserving mammalian cells using the present invention, the temperature can be arbitrarily selected as an appropriate temperature according to the type of cells to be preserved, but it is preferably within the range of 0 to 40°C. For example, when preserving platelets and megakaryocytes using the preservation solution of the present invention, the temperature is preferably 15 to 30°C, more preferably 20 to 24°C, and most preferably 21 to 23°C. Also, when preserving mesenchymal stem cells and T cells using the preservation solution of the present invention, the temperature is preferably 1 to 38°C, more preferably 1 to 30°C, particularly preferably 1 to 15°C, and most preferably 1 to 5°C. Furthermore, the temperature when preserving mammalian cells using the present invention can also be varied within the range of 0 to 40°C. For example, as shown in the following examples, when preserving T cells using the preservation solution of the present invention, after storing at a low temperature (for example, 1 to 5°C, preferably 5°C) for a certain period of time, it can be further stored at room temperature (for example, 20 to 26°C, preferably 22 to 25°C, more preferably 25°C) for several more hours. Also, the preservation solution of the present invention can be used to preserve mammalian cells for several hours to several tens of days. The preservation period varies depending on the type of cells to be preserved, but as shown in the following examples, for platelets and megakaryocytes, it can be 1 to 15 days, preferably 1 to 10 days; for mesenchymal stem cells, it can be 1 to 63 days, preferably 1 to 35 days, more preferably 1 to 30 days, more preferably 1 to 28 days, further preferably 1 to 14 days; for T cells, it can be 1 to 30 days, preferably 1 to 14 days, more preferably 1 to 2 days, and further preferably 30 hours.

[0035] The preservation solution of the present invention may be used for the use of administering mammalian cells to a mammal. That is, after storing a mixed solution containing the preservation solution of the present invention and mammalian cells under predetermined conditions, the mixed solution can be directly administered (for example, intravenous administration) into the living body of a mammal. Therefore, it is preferable that the preservation solution of the present invention does not contain components that can have an adverse effect on the living body of a mammal when administered into the living body of a mammal. Examples of such "components that can have an adverse effect" include polyvinylpyrrolidone, 2-mercaptoethanol, okadaic acid, sodium butyrate, G418, and the like.

[0036] As one aspect of the present invention, a mammalian deterioration inhibitor containing an essential protective component of the present invention can be mentioned. Such a deterioration inhibitor may further contain an optional protective component of the present invention. The mammalian deterioration inhibitor of the present invention can be used to prepare the preservation solution of the present invention by adding it to the above isotonic solution, and can also be used to enhance the preservability by adding it to a known mammalian preservation solution. The present invention also relates to a mammalian cell preservation container in which the preservation solution or powder preparation of the present invention is enclosed. The preservation container of the present invention may be in any form as long as it can maintain sterility after injecting a suspension of mammalian cells. Examples thereof include a blood bag, an infusion bag, a syringe, an ampoule, a vial, etc., and a blood bag is preferable.

[0037] Hereinafter, the present invention will be described more specifically by way of examples, but the technical scope of the present invention is not limited to these exemplifications.

Examples

[0038] [Preparation of iPS cell-derived platelets] iPS cell-derived platelets were prepared according to the method described in PCT / JP2018 / 034667. The specific procedure is shown in the following (1-1) to (1-12).

[0039] (1-1) Preparation of hematopoietic progenitor cells from iPS cells According to the method of Takayama et al. (J. Exp. Med., 2010, vol. 13, 2817 - 2830), differentiation culture from human iPS cells (TKDN SeV2 and NIH5: iPS cells derived from human fetal skin fibroblasts established using Sendai virus) into blood cells was performed. Specifically, human ES / iPS cell colonies were co-cultured with C3H10T1 / 2 feeder cells for 14 days in the presence of 20 ng / mL of VEGF (manufactured by R&D SYSTEMS) to produce hematopoietic progenitor cells (HPC). The above culture was carried out under the conditions of 37°C, 20% O2, and 5% CO2.

[0040] (1 - 2) Gene transfer system The gene transfer system utilized a lentiviral vector system. The lentiviral vector is a Tetracycline-regulated Tet-on (registered trademark) gene expression induction system vector. The mOKS cassette of LV-TRE-mOKS-Ubc-tTA-I2G (Kobayashi et al., Cell, 2010, vol. 142, No. 5, 787 - 799) was recombined into c-MYC, BMI1, or BCL-xL. The vectors into which c-MYC, BMI1, or BCL-xL was introduced were designated as LV-TRE-c-Myc-Ubc-tTA-I2G, LVTRE-BMI1-Ubc-tTA-I2G, and LV-TRE-BCL-xL-Ubc-tTA-I2G, respectively. The c-MYC, BMI1, and BCL-xL viruses were produced by introducing the above lentiviral vectors into 293T cells. By infecting the obtained viruses into the target cells, the c-MYC, BMI1, and BCL-xL genes are introduced into the genomic sequence of the target cells. These genes stably introduced into the genomic sequence can be forced to express by adding doxycycline (clontech#631311) to the medium.

[0041] (1 - 3) Infection of c-MYC and BMI1 viruses into hematopoietic progenitor cells On a 6-well plate seeded with C3H10T1 / 2 feeder cells in advance, the HPC obtained by the method of (1-1) above was seeded at 5×10 4 cells / well, and c-MYC and BMI1 were forcibly expressed by the lentivirus method using BMI1 virus and c-MYC virus. At this time, 6 wells were used for each cell line. Specifically, virus particles were added to the medium so that the multiplicity of infection (MOI) was 20, and infection was carried out by spin infection (centrifugation at 32 °C, 900 rpm, for 60 minutes). The above spin infection was carried out twice every 12 hours. The medium was IMDM (manufactured by Sigma-Aldrich) containing basic medium (15% Fetal Bovine Serum (manufactured by GIBCO), 1% Penicillin-Streptomycin-Glutamine (manufactured by GIBCO), 1% Insulin, Transferrin, Selenium Solution (ITS-G) (manufactured by GIBCO), 0.45 mmol / L 1-Thioglycerol (manufactured by Sigma-Aldrich), 50 μg / mL L-Ascorbic Acid (manufactured by Sigma-Aldrich)), to which 50 ng / mL Human thrombopoietin (TPO) (manufactured by R&D SYSTEMS), 50 ng / mL Human Stem Cell Factor (SCF) (manufactured by R&D SYSTEMS) and 2 μg / mL Doxycycline (DOX, manufactured by clontech, #631311) were added (hereinafter referred to as "differentiation medium"), and further, Protamine was added so that the final concentration was 10 μg / mL and used.

[0042] (1-4) Preparation and maintenance culture of megakaryocyte self-propagating strain Using the day when the infection with c-MYC and BMI1 viruses was carried out by the method of (1-3) above as day 0 of infection, megakaryocyte self-propagating strains were prepared by culturing HPCs into which the c-MYC gene and BMI1 gene were introduced as follows. Forced expression of the c-MYC gene and BMI1 gene was carried out by adding 1 μg / mL DOX to the medium. Day 2 to Day 11 of infection: On Day 2 of infection, the virus-infected blood cells obtained by the above method were collected by pipetting, centrifuged at 1200 rpm for 5 minutes to remove the supernatant, and then suspended in fresh differentiation medium and seeded on new C3H10T1 / 2 feeder cells (6-well plate). Passage was carried out by performing the same operation on Day 9 of infection. At the time of the above reseeding, after counting the cell number, it was seeded on C3H10T1 / 2 feeder cells (6-well plate) so that it became 1×10 5 cells / 2mL / well. Day 12 to Day 13 of infection: The same operation as on Day 2 of infection was carried out. After counting the cell number, it was seeded on C3H10T1 / 2 feeder cells (100-mm dish) so that it became 3×10 5 cells / 10mL / 100mm dish. Day 14 of infection: The virus-infected blood cells were collected, and 2 μL of anti-human CD41a-APC antibody (manufactured by BioLegend), 1 μL of anti-human CD42b-PE antibody (manufactured by eBioscience), and 1 μL of anti-human CD235ab-pacific blue antibody (manufactured by BioLegend) were used per 1.0×10 5 cells, respectively, to react the above blood cells with the antibodies. After the above reaction, analysis was carried out using FACS Verse (trademark) (manufactured by BD Biosciences). On Day 14 of infection, cells with a CD41a positive rate of 50% or more were used as megakaryocyte self-renewing strains.

[0043] (1-5) Lentiviral infection of the megakaryocyte self-renewing strain with BCL-xL virus BCL-xL was introduced into the megakaryocyte self-renewing strain on Day 14 of the above infection by the lentiviral method using BCL-xL virus. Virus particles were added to the medium so that MOI became 10, and infection was carried out by spin infection (centrifugation at 32 °C, 900 rpm, for 60 minutes). Forced expression of the BCL-xL gene was carried out by adding DOX to the medium so that it became 1 μg / mL DOX.

[0044] (1 - 6) Preparation and maintenance culture of immortalized megakaryocyte strains Day 14 to Day 18 after infection: The megakaryocyte self - proliferating strain transfected with the BCL - xL gene obtained by the method of (1 - 5) above was collected and centrifuged at 1200 rpm for 5 minutes. After the above centrifugation, the precipitated cells were suspended in a new differentiation medium and then seeded onto new C3H10T1 / 2 feeder cells at a density of 2×10 5 cells / 2mL / well (6 - well plate). Day 18 after infection (sub - culture): The megakaryocyte self - proliferating strain after transfection with the BCL - xL gene was collected. After counting the cell number, it was seeded at a density of 3×10 5 cells / 10mL / 100mm dish. Day 24 after infection (sub - culture): The megakaryocyte self - proliferating strain after transfection with the BCL - xL gene was collected. After counting the cell number, it was seeded at a density of 1×10 5 cells / 10mL / 100mm dish. Thereafter, sub - culture was performed in the same manner every 4 - 7 days for maintenance culture. At the time of sub - culture, after suspending in a new differentiation medium, it was seeded. The megakaryocyte self - proliferating strain transfected with BCL - xL on Day 24 after infection was collected. For each 1.0×10 5 cells, it was immunostained with 2μL of anti - human CD41a - APC antibody (manufactured by BioLegend), 1μL of anti - human CD42b - PE antibody (manufactured by eBioscience), and 1μL of anti - human CD235ab - Pacific Blue antibody (Anti - CD235ab - PB; manufactured by BioLegend), and then analyzed using FACS Verse (trademark). And on Day 24 after infection, the strains with a CD41a positive rate of 50% or more were used as immortalized megakaryocyte cell lines. These cells that could proliferate for 24 days or more after infection were designated as immortalized megakaryocyte cell lines SeV2 - MKCL and NIH5 - MKCL. The obtained SeV2 - MKCL and NIH5 - MKCL were statically cultured in a 10 - cm dish (10mL / dish). The medium was based on IMDM and the following components were added (concentrations are final concentrations). The culture conditions were 27℃, 5% CO 2 as described. FBS (manufactured by Sigma, #172012, lot.12E261) 15% L-Glutamin (manufactured by Gibco, #25030-081) 2 mmol / L ITS (manufactured by Gibco, #41400-045) Diluted 100-fold MTG (monothioglycerol, manufactured by sigma, #M6145-25ML) 450 μmol / L Ascorbic acid (manufactured by sigma, #A4544) 50 μg / mL Puromycin (manufactured by sigma, #P8833-100MG) 2 μg / mL SCF (manufactured by Wako Pure Chemical Industries, #193-15513) 50 ng / mL TPO-like substance 200 ng / mL

[0045] (1-7) Production of megakaryocyte cultures Forced expression was released by culturing in a medium without DOX. Specifically, the immortalized megakaryocyte cell lines (SeV2-MKCL and NIH5-MKCL) obtained by the method described in (1-6) above were washed twice with PBS(-) and suspended in the following platelet production medium. The seeding density of the cells was 1.0×10 5 cells / mL. Then, the cells were cultured for 6 days in the presence of the above platelet production medium to produce platelets, thereby producing megakaryocyte cultures. The above platelet production medium was based on IMDM and the following components were added (concentration is the final concentration). human plasma 5% L-Glutamin (manufactured by Gibco, #25030-081) 4 mmol / L ITS (manufactured by Gibco, #41400-045) Diluted 100-fold MTG (monothioglycerol, manufactured by sigma, #M6145-25ML) 450 μmol / L Ascorbic acid (manufactured by sigma, #A4544) 50 μg / mL SCF (manufactured by Wako Pure Chemical Industries, #193-15513) 50 ng / mL TPO-like substance 200 ng / mL ADAM inhibitor, 15 μmol / L GNF351 (manufactured by Calbiochem, #182707), 500 nmol / L Y39983 (manufactured by Chemscene LLC, #CS - 0096), 500 nmol / L Urokinase, 5 U / mL Low molecular weight heparin (manufactured by SANOFI, Clexane), 1 U / mL

[0046] Concentration of the culture of (1 - 8) megakaryocytes Platelets were produced (purified) from the culture of megakaryocytes obtained in (1 - 7) above. Note that the same purification was carried out twice. Specifically, the culture of megakaryocytes obtained in (1 - 7) above was introduced into a culture bag. Then, the culture bag was connected to a concentration system as shown in Figure 1. In Figure 1, the washing and storage solution bags 1 and 2 contain the washing and storage solution. The above washing and storage solution was prepared by adding 20% ACD and 2.5% human serum albumin to a bicarbonate infusion solution (manufactured by Otsuka Pharmaceutical Co., Ltd.) and adjusting the pH to 7.2 with NaOH. Then, according to Table 1 below, using a hollow fiber membrane (Plasmaflow OP, manufactured by Asahi Kasei Medical Co., Ltd.), the culture of megakaryocytes was concentrated, and the concentrated solution of the culture of megakaryocytes obtained was collected in a storage bag.

[0047]

Table 1

[0048] (1 - 9) Centrifugation of the culture First, using a sterile connection device, the waste liquid bag of the ACP215 disposable set was replaced with a collection bag. The above collection bag used a high - calic IVH bag (manufactured by Terumo, HC - B3006A). Next, 10% of the ACD - A solution (manufactured by Terumo) was added to the concentrated solution of the culture of megakaryocytes. After the above addition, the concentrated solution to which the ACD - A solution was added was injected into a cell bag. The above cell bag used a high - calic IVH bag (manufactured by Terumo, HC - B3006A). Furthermore, using a sterile joining device, a cell bag containing a culture supplemented with ACD-A solution was joined to an ACP215 disposable set. Then, the ACP215 was started in service mode and the rotation speed was set to 2500 rpm (350×g). The ACP215 was started, and the culture in the above cell bag was introduced into the separation bowl at about 100 mL / min. The liquid component flowing out from the above separation bowl was collected in a collection bag. After introducing the entire amount of the culture in the above cell bag into the separation bowl, an additional 500 mL of washing and preservation solution was introduced into the separation bowl. After introducing the washing and preservation solution into the separation bowl, the centrifugation was stopped and the collection bag containing the recovered liquid (the recovered liquid component containing platelets) was detached using a tube sealer. A collection bag containing the recovered liquid (containing platelets) was joined to a new ACP215 disposable set using the above sterile joining device. The ACP215 was started in normal mode. The program setting was to select WPC, and according to the instructions of the device, the ACP215 disposable set joined with the above collection bag was set. Note that the collection bag containing the recovered liquid was placed on a stand. Next, the centrifugation speed of the ACP215 was changed to 5000 rpm (1398.8×g) and the centrifugation was started. When the recovered liquid started to be introduced into the above separation bowl, the injection was changed from automatic to manual. Specifically, the recovered liquid was introduced into the separation bowl at an introduction speed of about 100 mL / min. After adding the entire amount of the recovered liquid to the separation bowl, an additional 500 mL of washing and preservation solution was added.

[0049] (1-10) Washing of the culture The washing was performed with 2000 mL of the above washing and preservation solution according to the program of the ACP215.

[0050] (1-11) Recovery of the culture According to the program of the ACP215, 200 mL of the washed culture (containing platelets) was recovered into a platelet preparation bag.

[0051] (1-12) Separation of the culture Regarding the above platelet preparation bag, platelets were separated by a conventional method using the above hollow fiber membrane and collected in a collection bag.

Example

[0052] [Addition of Vitamin C to Platelet Preservation Solution] (2-1) Preparation of Platelet Preservation Solution A solution was prepared by adding a human serum albumin preparation (HSA; manufactured by CSL Behring) and a blood preservation solution (ACD-A solution; manufactured by Terumo) (sodium citrate hydrate 2.20 W / V%, citric acid hydrate 0.80 W / V%, and glucose 2.20 W / V%) to Ringer's bicarbonate solution (Bicarb Infusion; manufactured by Otsuka Pharmaceutical Factory) (sodium chloride 5.84 g / L, potassium chloride 0.30 g / L, calcium chloride hydrate 0.22 g / L, magnesium chloride 0.20 g / L, sodium bicarbonate 2.35 g / L, and sodium citrate hydrate 0.20 g / L). In this specification, this solution is also referred to as the "first-generation preservation solution". Further, a solution was prepared by adding a VC preparation (an injection preparation containing additives; manufactured by Sawai Pharmaceutical) to the first-generation preservation solution. In this specification, this solution is also referred to as the "VC-added preservation solution". The final concentrations of the above additives in each preservation solution are shown in Table 2 below. Also, each preservation solution was adjusted to pH 7.3 ± 0.1 with 1 M NaOH and incubated for 1 hour or more until use (under light shielding, at room temperature, 5% CO 2 ).

[0053]

Table 2

[0054] (2-2) Preparation of Platelet Samples Using Each Preservation Solution The concentration of platelets contained in the culture obtained in Example 1 was measured by FACS. A culture containing the required amount of platelets was aliquoted, and ACD-A solution (10 v / v%) and PEG1 (final concentration 2 μM; manufactured by Cayman Chemical Company) were added, followed by centrifugation for 12 minutes (1200×g, 22 °C). After removing the supernatant, the storage solution prepared in (2-1) above was added to the pellet and gently suspended to obtain a uniform suspension (platelet concentration: about 0.3×10 9 plts / mL). Each suspension was seeded in a 24-well plate and stored with horizontal shaking for up to 5 days (under light shielding, 22 °C, 50 rpm), and then used for the experiments in (2-3) and (2-4) below.

[0055] (2-3) Annexin V positive rate of platelet samples Annexin V is one of the indicators of platelet deterioration (activation). When the positive rate is high, it is evaluated that the platelets are deteriorated or abnormal. Therefore, the Annexin V positive rate in the platelet samples obtained in (2-2) above was measured, and the effect of suppressing platelet deterioration by each storage solution was examined. Specifically, the platelet samples obtained in (2-2) above were diluted 500-fold with Annexin Buffer (manufactured by Beckton Dickinson) and dispensed into three centrifuge tubes (negative control, positive control, and unstimulated sample, respectively). EDTA was added to the negative control sample and Ionomycin was added to the positive control sample. Then, all samples were stained with anti-CD41 antibody (manufactured by BioLegend) and Annexin V (manufactured by Beckton Dickinson) (under light shielding, room temperature, 20 minutes). After staining, Annexin Buffer was added and immediately measured by FACS. The Annexin V positive rate in the iPS platelets (CD41 + fraction) in the negative control was set to 1.0 ± 0.1%, and the Annexin V positive rate in the unstimulated sample was calculated. The results are shown in the upper part of Figure 2 (all are the results obtained from the platelet samples after 5-day storage). Platelets (CD41 +The Annexin V positive rate of the sub-drawings) was 60.5±0.3 to 64.4±0.1% when the first-generation preservation solution was used, while it was 53.9±2.1 to 56.7±1.8% when the VC-added preservation solution was used, indicating that it decreased with the addition of VC. From these results, it was clarified that the platelet deterioration inhibitory effect of the platelet preservation solution was improved by adding 300 to 3000 mg / L of VC.

[0056] (2-4) P-Selectin positive rate of platelet samples without stimulation and PAC-1 / P-Selectin positive rate upon ATR stimulation P-Selectin is one of the indicators of platelet deterioration (activation). When the P-Selectin positive rate without stimulation is high, the platelets are evaluated as deteriorated or abnormal. Therefore, the P-Selectin positive rate in the platelet samples obtained in (2-2) above was measured to examine the platelet deterioration inhibitory effect of each preservation solution. In addition, to know the preservation state of platelet function, the PAC-1 and P-Selectin positive rates were examined when each platelet sample was stimulated with adenosine diphosphate (ADP) and thrombin receptor activating peptide-6 (TRAP-6), which are platelet activators (hereinafter, the combination of ADP and TRAP-6 may be referred to as "ATR"). Specifically, the platelet sample obtained by the above (2-2) was diluted 500-fold with Tyroad HEPES Buffer (THB) and dispensed into three centrifuge tubes (negative control, positive control, and unstimulated sample, respectively). A mixed solution of ADP (manufactured by Sigma, final concentration 20 μM) and TRAP-6 (manufactured by BACHEM, final concentration 30 μM) was added to the positive control sample. The positive control sample and the unstimulated sample after stimulation were stained with anti-CD41 antibody (manufactured by BioLegend), anti-P-Selectin antibody (manufactured by BioLegend), and anti-PAC-1 antibody (manufactured by Beckton Dickinson) (under light shielding, at room temperature, for 30 minutes). The negative control sample was stained with anti-CD41 antibody (manufactured by BioLegend), isotype control antibody of anti-P-Selectin antibody (manufactured by BioLegend), and isotype control antibody of anti-PAC-1 antibody (manufactured by BioLegend) (under light shielding, at room temperature, for 30 minutes). 1% paraformaldehyde was added to the stained sample for fixation (under light shielding, at 4°C, for 30 minutes or more), and then the positive rate of P-Selectin and / or PAC-1 was measured by FACS within 24 hours. At the time of analysis, gating was performed so that the positive rates of P-Selectin and PAC-1 in the iPS platelets (CD41 + fraction) in the negative control sample were 1.0 ± 0.1% or less.

[0057] The positive rate of P-Selectin without stimulation is shown in the middle section of FIG. 2, and the positive rate of PAC-1 / P-Selectin upon ATR stimulation is shown in the lower section of FIG. 2 (both are results obtained from the platelet sample after storage for 5 days). Unstimulated platelets (CD41 +The P-Selectin positive rate in the platelet fraction was 33.2±0.3 to 45.6±1.2% when the first-generation preservation solution was used, whereas it was 13.0±0.2 to 19.7±1.1% when the VC-added preservation solution was used, indicating that it decreased with the addition of VC. Also, a tendency for the decrease in the P-Selectin positive rate to depend on the added VC concentration was observed. From these results, it was shown that by adding 300 to 3000 mg / L of VC, the inhibitory effect on platelet deterioration in the platelet preservation solution was improved. Also, the platelets after ATR stimulation (CD41 + The PAC-1 / P-Selectin positive rate in the platelet fraction was 17.6 to 20.8% when the first-generation preservation solution was used, whereas it was 23.9 to 26.9% when the VC-added preservation solution was used, indicating that it increased with the addition of VC. From these results, it was shown that by adding 300 to 3000 mg / L of VC, the effect of maintaining platelet function in the platelet preservation solution was improved.

Example

[0058] [Addition of VC and VB3 to Platelet Preservation Solution] (3-1) Preparation of Platelet Preservation Solution A solution was prepared by adding nicotinic acid (nicotinic acid injection; manufactured by Toa Aiyo Co., Ltd.) to the VC-added preservation solution. As described above, in this specification, "VB3" means nicotinic acid and / or nicotinamide. However, in Examples 3 to 5 and 8 to 13, nicotinic acid was used as VB3, and in Example 6, nicotinic acid or nicotinamide was used as VB3. Also, in this specification, a solution obtained by adding VB3 (nicotinic acid or nicotinamide) to the VC-added preservation solution is also referred to as "VC / VB3-added preservation solution" or "second-generation preservation solution". The final concentrations of the above additives in each preservation solution are as shown in Table 3 below. Also, each preservation solution was adjusted to pH 7.3±0.1 with 1M NaOH and incubated for 1 hour or more until use (under light shielding, at room temperature, 5% CO 2 ).

[0059]

Table 3

[0060] (3-2) Preparation of platelet samples using each storage solution The storage solution prepared in the above (3-1) was added to the iPS cell-derived platelet preparation prepared by the method described in Example 1, and gently suspended to form a uniform suspension (platelet concentration: about 0.3×10 9 plts / mL). Each suspension was immediately, or seeded in a 24-well plate and horizontally shaken and stored for up to 5 days (under light shielding, 22 degrees, 50 rpm), and then subjected to the following experiments (3-3) to (3-5).

[0061] (3-3) Annexin V positive rate of platelet samples The Annexin V positive rate in the platelet samples obtained in the above (3-2) (after 5-day storage) was measured by the method described in (2-3) of Example 2, and the inhibitory effect of each storage solution on platelet deterioration was examined. The results are shown in Figure 3. The Annexin V positive rate of platelets (CD41 + fraction) was 61.4±1.9% when using the first-generation storage solution, 46.3±0.7% when using the VC-added storage solution, and 44.3±0.5% when using the VC / VB3-added storage solution. From these results, it became clear that by using a combination of VC and VB3, platelet deterioration was more strongly suppressed compared to using only VC.

[0062] (3-4) Lactic acid production of platelet samples In platelet preparations during storage, lactic acid is produced by anaerobic metabolism. It is known that as the lactic acid concentration increases, the pH decreases, and as a result, platelet deterioration occurs. Therefore, the lactic acid concentration in the platelet samples obtained in the above (3-2) was measured, and the inhibitory effect of each storage solution on lactic acid production was examined. Specifically, the platelet sample obtained by the above (3-2) (after 5-day storage) was placed in a 1.5 mL tube and centrifuged (1200×g, 22°C, 10 minutes). The supernatant was collected and placed in a new tube, and stored frozen at -80°C until measurement. The lactic acid concentration in the above supernatant was measured using an N-assay L LAC Nyttobo (manufactured by Nyttobo Medical Co., Ltd.) and an automatic analyzer 7180 (manufactured by Hitachi High-Technologies Corporation). The results are shown in Figure 4. The lactic acid concentration in the sample supernatant was 0.35±0.01 g / L when using the first-generation preservation solution, 0.16±0.01 g / L when using the VC-added preservation solution, and 0.13±0.01 g / L when using the VC / VB3-added preservation solution. From these results, it became clear that by combining and using VC and VB3, anaerobic metabolism in platelets during storage was more strongly suppressed compared to using only VC.

[0063] (3-5) Platelet recovery rate of platelet samples Activation of platelets during storage is thought to cause adhesion to the container and formation of aggregates, leading to a decrease in platelet recovery rate. Therefore, the platelet concentrations in the platelet samples before and after storage obtained by the above (3-2) were measured, and the change in the recovery rate by each preservation solution was examined. Specifically, the platelet samples prepared in the above (3-2) (before storage and after 5-day storage) were diluted 500-fold with THB and dispensed into TruCOUNT tubes (manufactured by Beckton Dickinson). Stained with anti-CD41 antibody (manufactured by BioLegend) and anti-CD42b antibody (manufactured by BioLegend) (under light shielding, room temperature, 20 minutes), and after adding THB again, measured by FACS. Based on the bead count value of the TruCOUNT tube, the concentration of CD41 + cells (platelets) was calculated. For each platelet sample, the recovery rate was calculated with the platelet concentration before storage as the denominator and the platelet concentration after storage as the numerator. The results are shown in Fig. 5. The platelet recovery rate was 102.0 ± 4.2% when using the first-generation preservation solution, 101.1 ± 6.8% when using the VC-added preservation solution, and 99.9 ± 3.9% when using the VC / VB3-added preservation solution. From these results, it became clear that even when VC and / or VB3 were added, no difference was observed in the recovery rate.

Example

[0064] [Addition of VC and nicotinic acid to platelet preservation solution] (4-1) Preparation of platelet preservation solution and preparation of platelet samples A VC preparation (1000 mg / L, manufactured by Sawai Pharmaceutical Co., Ltd.) was added to the first-generation preservation solution (containing 20% ACD-A solution) to prepare a VC-added preservation solution. Further, nicotinic acid (400 mg / L, manufactured by Toa Aiyo Co., Ltd.) was added to the VC-added preservation solution to prepare a second-generation preservation solution. The pH of each solution was adjusted to 7.3 ± 0.1. Table 4 below shows the composition of the second-generation preservation solution used in this example.

[0065]

Table 4

[0066] The above preservation solution (first-generation preservation solution, VC-added preservation solution, or second-generation preservation solution) was added to the iPS cell-derived platelet preparation prepared by the method described in Example 1, and gently suspended to form a uniform suspension (platelet concentration: approximately 1.0×10 9 plts / mL). Each suspension was immediately, or filled into a blood storage bag and horizontally shaken and stored for 5 or 10 days (under light shielding, 22 °C, 50 rpm), and then used in the following experiments (4-2) to (4-4).

[0067] (4-2) Lactate concentration and pH of platelet samples As described in (2-4) above, lactic acid is produced by anaerobic metabolism in platelet preparations during storage, and it is known that this causes a decrease in pH. In this experiment, the lactic acid concentration and pH in the platelet samples obtained by (4-1) above were measured, and the inhibitory effect on lactic acid production by each storage solution was examined. Specifically, the platelet samples obtained by (4-1) above (before storage, after 5 days of storage, and after 10 days of storage) were placed in 1.5 mL tubes, and the lactic acid concentration and pH were measured using a cell culture analyzer FLEX (manufactured by Nova Biomedical). The results are shown in Fig. 6. As shown in the left graph of Fig. 6, the lactic acid concentration increased over time regardless of the storage solution used, but the degree of increase was the lowest in the samples using the second-generation storage solution. Also, as shown in the right graph of Fig. 6, the pH decreased over time regardless of the storage solution used, but the degree of decrease was the lowest in the samples using the second-generation storage solution, and it remained neutral even after 10 days of storage.

[0068] (4-3) P-Selectin positive rate of platelet samples without stimulation and PAC-1 / P-Selectin positive rate at the time of ATR stimulation As described in (2-4) of Example 2, P-Selectin is known as a platelet degradation marker, and PAC-1 / P-Selectin at the time of ATR (ADP / TRAP-6) stimulation is known as a platelet reactivity marker. Therefore, in this experiment, by the method described in (2-4) of Example 2, the P-Selectin positive rate of the platelet samples obtained by (4-1) above without stimulation and the PAC-1 / P-Selectin positive rate at the time of ATR stimulation were measured, and the inhibitory effect on platelet degradation by each storage solution was examined. The non-stimulated P-Selectin positive rate is shown on the left of Figure 7, and the PAC-1 / P-Selectin positive rate upon ATR stimulation is shown on the right of Figure 7. The P-Selectin positive rate was 18.0% before storage (Day1) when using the first-generation preservation solution, but increased to 33.0% after 5-day storage (Day5) and 31.1% after 10-day storage (Day10). On the other hand, when using the VC-added preservation solution, the P-Selectin positive rates after 5 and 10 days of storage were both lower compared to when using the first-generation preservation solution, being 26.2% (Day5) and 24.4% (Day10) respectively. Also, when using the second-generation preservation solution, the P-Selectin positive rates after 5 and 10 days of storage were even lower than when using the VC-added preservation solution, being 24.2% (Day5) and 19.3% (Day10) respectively. From the above results, it became clear that by using the second-generation preservation solution, platelet deterioration is suppressed even after 10-day storage. Also, the ATR-stimulated PAC-1 / P-Selectin positive rate was 43.4% before storage (Day1) when using the first-generation preservation solution, but decreased rapidly to 34.5% after 5-day storage (Day5) and 5.9% after 10-day storage (Day10). On the other hand, when using the VC-added preservation solution or the second-generation preservation solution, the ATR-stimulated PAC-1 / P-Selectin positive rates were comparable to those when using the first-generation preservation solution after 5-day storage (Day5) (35.9% and 35.3% respectively), but showed significantly higher values than the first-generation preservation solution after 10-day storage (Day10) (19.4% and 19.9% respectively). From the above results, it became clear that by using the VC-added preservation solution or the second-generation preservation solution, platelet reactivity is maintained even after 10-day storage.

[0069] (4-4) Platelet recovery rate of platelet samples The platelet concentration and recovery rate in the platelet samples obtained by the method described in (3-5) above were measured. The results are shown in Fig. 8. The platelet recovery rates when using the first-generation storage solution were 85.4% after 5-day storage (Day5) and 86.8% after 10-day storage (Day10). On the other hand, when using the VC-added storage solution or the second-generation storage solution, the platelet recovery rates were over 90% even after 10-day storage (90.0% and 91.4% respectively). These results are consistent with the result of (4-3) above, that is, the platelet activation during storage is suppressed by using the VC-added storage solution or the second-generation storage solution.

Example

[0070] [Aggregation ability of platelet samples] (5-1) Preparation of storage solution and platelet samples The first-generation storage solution and the second-generation storage solution were prepared by the method described in (3-1) of Example 3. The above storage solution was added to the iPS cell-derived platelet preparation prepared by the method described in Example 1 and gently suspended to form a uniform suspension (platelet concentration: about 1.0×10 9 plts / mL). Each suspension was immediately, or filled into a blood storage bag and horizontally shaken and stored for 5 or 10 days (under light shielding, 22 degrees, 50 rpm), and then used in the experiment of (5-2) below.

[0071] (5-2) Measurement of aggregation ability of platelet samples The platelet samples obtained by (5-1) above were centrifuged (1200×g, room temperature, 10 minutes), and the supernatant was removed. Ringer's bicarbonate solution containing 5% ACD-A was added and suspended to a cell concentration of 1.0×10 9 plts / ml. The obtained suspension was diluted with human plasma / CaCl 2 solution (manufactured by Cosmo Bio Co., Ltd.) and stimulated by adding the stimulant shown in Table 5 below. The aggregation rate of each sample after stimulation was measured by a platelet aggregation ability measuring device PRP313M (manufactured by Taiyo Corporation).

[0072]

Table 5

[0073] Figures 9 to 12 respectively show the results obtained using TRAP-6, Collagen, ADP, and Collagen / ADP as stimulants. It was revealed that the maximum aggregation rate of platelet samples using the first-generation preservation solution significantly decreased during storage for 5 to 10 days regardless of the stimulant used (''Conventional preservation solution (Day 10)'' in Figures 9 to 12). On the other hand, such a sharp decrease in the aggregation rate was not observed in platelet samples using the second-generation preservation solution (''New preservation solution (Day 10)'' in Figures 9 to 12). From the above results, it became clear that by using the second-generation preservation solution, the aggregating ability (hemostatic ability) of platelets during storage can be maintained for 10 days or more.

Example

[0074] [Addition of nicotinic acid or nicotinamide to the preservation solution] (6-1) Preparation of preservation solution and platelet sample To the first-generation preservation solution (containing 20% ACD-A solution), VC (1000 mg / L) and nicotinic acid (400 mg / L; manufactured by Fujifilm Wako Pure Chemical Corporation) or nicotinamide (400 mg / L; manufactured by Fujifilm Wako Pure Chemical Corporation) were added and adjusted to pH 7.3 ± 0.1 using 1M NaOH. Hereinafter, the preservation solution obtained by adding VC and nicotinic acid to the first-generation preservation solution may be referred to as ''second-generation preservation solution (nicotinic acid)'', and the preservation solution obtained by adding VC and nicotinamide to the first-generation preservation solution may be referred to as ''second-generation preservation solution (nicotinamide)''. The above preservation solution was added to the iPS cell-derived platelet preparation prepared by the method described in Example 1 and gently suspended to obtain a uniform suspension (platelet concentration: about 0.3×10 9 plts / mL). Each suspension was immediately used, or seeded in a 24-well plate and horizontally shaken and stored for 5 days (under light shielding, 22 degrees, 50 rpm), and then used in the following experiments (6-2) and (6-3).

[0075] (6-2) Change in the Annexin V positive rate of platelet samples By the method described in (2-3) of Example 2, the positive rate of Annexin V (degradation marker) in the platelet sample obtained by the above (6-1) was measured, and the effect of each preservation solution on suppressing platelet degradation was examined. The results are shown in the upper part of Fig. 13. In the figure, "Day1" indicates the sample before preservation, and "First generation", "Second generation (nicotinic acid)", and "Second generation (nicotinamide)" indicate the samples after 5-day preservation, respectively. Platelets (CD41 + fraction) had an Annexin V positive rate of 38.2 ± 0.3% in the preservation solution added with nicotinic acid and 38.5 ± 0.3% in the preservation solution added with nicotinamide. These values were both lower than those when using the first-generation preservation solution (53.1 ± 0.4%). From these results, it became clear that even with vitamin B3 (nicotinic acid and / or nicotinamide) widely used, the degradation of platelets during preservation was suppressed.

[0076] (6-3) Influence of each preservation solution on the P-Selectin positive rate of the platelet sample without stimulation and the PAC-1 / P-Selectin positive rate during ATR stimulation By the method described in (2-4) of Example 2, the P-Selectin (degradation marker) positive rate of the platelet sample obtained by the above (6-1) without stimulation and the PAC-1 / P-Selectin (reactivity marker) positive rate during ATR stimulation were measured, and the effect of each preservation solution on suppressing platelet degradation was examined. The P-Selectin positive rate without stimulation is shown in the middle part of Fig. 13, and the PAC-1 / P-Selectin positive rate during ATR stimulation is shown in the lower part of Fig. 13. The P-Selectin positive rate increased from 11.6% before preservation (Day1) to 28.3 ± 0.5% after 5-day preservation (Day5) when using the first-generation preservation solution. On the other hand, when using the second-generation preservation solution added with nicotinic acid or nicotinamide, it was kept low at 9.4 ± 0.2% - 10.6 ± 0.2% even after 5-day preservation. In addition, the positive rate of ATR-stimulated PAC-1 / P-selectin was 33.9 ± 2.4% when using the first-generation preservation solution, but it was 41.6 ± 0.8% to 41.2 ± 1.9% when using the second-generation preservation solution added with nicotinic acid or nicotinamide. From these results, it became clear that the reactivity of platelets during storage was maintained by nicotinic acid or nicotinamide.

Example

[0077] [Addition of VC alone to the preservation solution] (7-1) Preparation of preservation solution and platelet sample In Examples 2 to 6 above, a VC preparation manufactured by Sawai Pharmaceutical Co., Ltd. was used as "VC". Since the VC preparation contains additives consisting of sodium pyrosulfite, L-cysteine hydrochloride monohydrate, and benzyl alcohol, it was confirmed by the following experiment whether these additives affected the experimental results. A VC reagent (VC without additives; 1000 mg / L, manufactured by Fujifilm Wako Pure Chemical Corporation) was added to the first-generation preservation solution (containing 20% ACD-A solution), and the pH was adjusted to 7.3 ± 0.1. Hereinafter, such a preservation solution may be referred to as the "first-generation preservation solution (VC reagent)". The above preservation solution was added to the iPS cell-derived platelet preparation prepared by the method described in Example 1, and gently suspended to form a uniform suspension (platelet concentration: about 0.3×10 9 plts / mL). Each suspension was immediately, or seeded in a 24-well plate and horizontally shaken and stored for 5 days (under light shielding, 22 degrees, 50 rpm), and then used for the following experiments (7-2) and (7-3).

[0078] (7-2) Positive rate of Annexin V of platelet sample According to the method described in (2-3) of Example 2, the positive rate of Annexin V (degradation marker) in the platelet sample obtained in the above (7-1) was measured, and the effect of suppressing platelet degradation by each preservation solution was examined. The results are shown in the upper part of Figure 14 (Day1 shows the sample before storage, and the first-generation and first-generation VC reagent show the samples after 5-day storage respectively). Platelets (CD41+ The Annexin V positive rate of the sub-drawing) was lower when using the first-generation VC reagent (38.9 ± 0.4%) than when using the first-generation preservation solution (53.1 ± 0.4%). From these results, it became clear that the addition of only VC (without VB3) suppresses the deterioration of iPS-derived platelets during storage.

[0079] (7-3) P-Selectin positive rate of platelet samples without stimulation and PAC-1 / P-Selectin positive rate upon ATR stimulation By the method described in (2-4) of Example 2, the P-Selectin (deterioration marker) positive rate of the platelet samples obtained in the above (7-1) without stimulation and the PAC-1 / P-Selectin (reactivity marker) positive rate upon ATR stimulation were measured to examine the effect of suppressing platelet deterioration by each preservation solution. The P-Selectin positive rate without stimulation is shown in the middle of FIG. 14, and the PAC-1 / P-Selectin positive rate upon ATR stimulation is shown in the lower part of FIG. 14. The P-Selectin positive rate without stimulation was lower when using the first-generation VC reagent (9.5 ± 0.4%) than when using the first-generation preservation solution (28.3 ± 0.5%). From these results, it became clear that the addition of only VC suppresses the deterioration of iPS-derived platelets during storage. Also, the PAC-1 / P-Selectin positive rate upon ATR stimulation was higher when using the first-generation VC reagent (38.2 ± 1.6%) than when using the first-generation preservation solution (33.9 ± 2.4%). From these results, it became clear that the addition of only the VC reagent maintains the reactivity of iPS-derived platelets during storage. That is, it became clear that the effect of adding VC is not due to the additives contained in the VC preparation manufactured by Sawai Pharmaceutical Co., Ltd.

Example

[0080] [Comparison with Japan Red Cross platelet washing solution] (8-1) Preparation of preservation solution and platelet samples Additives shown in Table 6 below were added to the Bikanet solution to prepare a second-generation storage solution. Also, a solution with the same composition as a known platelet storage solution (Japanese Red Cross Society, attached document of irradiated washed platelets - LR "Nichi Red", March 2016, and Japanese Journal of Transfusion and Cell Therapy, Vol. 59.No. 3 59(3):492―498, 2013) was prepared. In this specification, the known storage solution is also referred to as the "Nichi Red platelet washing solution". Each storage solution was adjusted to pH 7.3 ± 0.1 with 1 M NaOH and incubated for 1 hour or more until use (under light shielding, room temperature, 5% CO 2 ).). The above storage solution was added to the iPS cell-derived platelet preparation produced by the method described in Example 1 and gently suspended to form a uniform suspension (platelet concentration: about 1.0×10 9 plts / mL). Each suspension was immediately or filled into a blood storage bag and horizontally shaken and stored for 5 days (under light shielding, 22 degrees, 50 rpm), and then used in the following experiments (8-2) and (8-3).

[0081]

Table 6

[0082] (8-2) Annexin V positive rate of platelet samples By the method described in (2-3) of Example 2, the Annexin V (degradation marker) positive rate in the platelet samples obtained in (8-1) above was measured, and the effect of suppressing the degradation of platelets by each storage solution was examined. The results are shown in the upper part of the upper part of Figure 15 ("Day1" indicates the sample before storage, and "Day5" indicates the sample after 5-day storage). The Annexin V positive rate of platelets (CD41 + fraction) was lower when using the second-generation storage solution (49.3%) than when using the Nichi Red platelet washing solution (73.9%). From these results, it became clear that the second-generation storage solution suppresses the degradation of iPS-derived platelets during storage.

[0083] (8-3) P-Selectin positive rate of platelet sample without stimulation and PAC-1 / P-Selectin positive rate upon ATR stimulation By the method described in (2-4) of Example 2, the P-Selectin (degradation marker) positive rate without stimulation and the PAC-1 / P-Selectin (reactivity marker) positive rate upon ATR stimulation in the platelet sample obtained by the above (8-1) were measured, and the effect of inhibiting platelet degradation by each preservation solution was examined. The P-Selectin positive rate without stimulation is shown in the middle of FIG. 15, and the PAC-1 / P-Selectin positive rate upon ATR stimulation is shown in the lower part of FIG. 15. The P-Selectin positive rate without stimulation was lower when using the second-generation preservation solution (16.5%) than when using the Japan Red Cross platelet washing solution (30.4%). From these results, it became clear that in the preservation of iPS-derived platelets, the second-generation preservation solution has a better effect of inhibiting degradation than the Japan Red Cross platelet washing solution. Also, the PAC-1 / P-Selectin positive rate upon ATR stimulation was higher when using the second-generation preservation solution (24.8%) than when using the Japan Red Cross platelet washing solution (9.8%). From these results, it became clear that in the preservation of iPS-derived platelets, the second-generation preservation solution has a better effect of maintaining function than the Japan Red Cross platelet washing solution.

Example

[0084] [Hemostasis test using thrombocytopenic model mice] (9-1) Preparation of platelet sample The second-generation preservation solution was added to the iPS cell-derived platelet preparation prepared by the method described in Example 1, and gently suspended to form a uniform suspension (platelet concentration: about 1.0×10 9 plts / mL). Each suspension was immediately used for the following experiment (9-2), or filled into a blood storage bag and horizontally shaken and stored (under light shielding, 22 degrees, 50 rpm, for 10 days).

[0085] (9-2) Hemostasis test The platelet sample obtained by the above (9-1) was administered into the tail vein of thrombocytopenia model NOG mice (200 μL (2×10 8 plts) per mouse). Ten minutes after the administration, an incision was made in the ventral tail artery using an injection needle. One incision was made per individual. After confirming bleeding from the incision, the distal end of the tail including the incision site was immersed in physiological saline at 37°C, and the time until hemostasis was measured. The measurement time was up to 600 seconds. Also, as a control group (Vehicle), the same test was conducted using only the second-generation preservation solution (not containing platelets). The results are shown in Fig. 16. In the Vehicle-administered group, hemostasis within 600 seconds was not observed in all mice. On the other hand, in the platelet-administered group, the average time until hemostasis was 391 seconds, and the shortest individual was 130 seconds. From these results, it was clarified that iPS cell-derived platelets preserved by the second-generation preservation solution of the present invention have a hemostatic effect.

Example

[0086] [Preservation of human mesenchymal stem cells] (10-1) Preparation of mesenchymal stem cell preservation solution 3% trehalose and 5% dextran were added to lactated Ringer's solution (Lactec infusion; manufactured by Otsuka Pharmaceutical Factory, Inc.) (hereinafter, such a solution may be referred to as "CSP-01 solution". See JP 2012-115253 and WO2014 / 208053). To the above CSP-01 solution, a VC preparation (1000 mg / L, manufactured by Sawai Pharmaceutical Co., Ltd.) and nicotinic acid (400 mg / L, manufactured by Toa Aiyo Co., Ltd.) were added to prepare a mesenchymal stem cell preservation solution. Also, a control preservation solution was prepared by adding distilled water (Otsuka distilled water; manufactured by Otsuka Pharmaceutical Factory, Inc.) to the CSP-01 solution instead of VC and nicotinic acid.

[0087] (10-2) Preservation of mesenchymal stem cells Human bone marrow-derived mesenchymal stem cells (manufactured by Lonza) were suspended using the preservation solution prepared in the above (10-1) (5×10 5(cells / mL). After allowing the suspension to stand at 5°C for 24, 48, 96, and 168 hours, the total cell count and the number of dead cells were measured using a microscope. The cell viability (%) and viable cell recovery rate (%) at each time point were calculated using the following formulas 1 and 2.

[0088] [Formula 1] Cell viability (%) = (Total cell count - Number of dead cells) / Total cell count × 100

[0089] [Formula 2] Viable cell recovery rate (%) = Number of viable cells at each time point / Number of viable cells immediately after suspension (before storage) × 100

[0090] The results are shown in Fig. 17. When using the control preservation solution, it was revealed that the cell viability and viable cell recovery rate decreased rapidly after 48 hours of storage. On the other hand, when using the preservation solutions added with VC and nicotinic acid, no significant decrease in the cell viability and viable cell recovery rate was observed, and it was revealed that they were maintained at the same level as before storage even after 168 hours. From these results, it was shown that VC and nicotinic acid also have excellent effects in the preservation of mesenchymal stem cells.

Example

[0091] [Preservation of iPS cell-derived megakaryocytes] (11-1) Preparation of iPS cell-derived megakaryocytes The concentration of platelets contained in the culture obtained in Example 1 was measured by FACS. An appropriate amount of the culture was aliquoted, and ACD-A solution (10 v / v%) and PEG1 (final concentration 2 μM; manufactured by Cayman Chemical Company) were added, followed by centrifugation for 12 minutes (1200 × g, 22°C, minimum brake). After removing the supernatant, the first-generation or second-generation preservation solution was added to the pellet and gently suspended to form a uniform suspension (platelet concentration: approximately 1.3 × 10 9 plts / mL). Each suspension was immediately used or filled into a blood storage bag and horizontally shaken and stored for 5 or 10 days (under light shielding, 22 degrees, 50 rpm), and then subjected to the experiment in (11-2) below.

[0092] (11-2) Annexin V negative rate of megakaryocyte sample Annexin V is also known as a probe for detecting changes in the cell membrane (exposure of phosphatidylserine to the outside of the cell membrane) in apoptotic cells. Therefore, the Annexin V negative rate in the megakaryocyte sample obtained by the above (11-1) was measured, and the apoptosis inhibitory effect of each preservation solution on megakaryocytes was examined. Specifically, the megakaryocyte sample (culture containing megakaryocytes and platelets) obtained by the above (11-1) was diluted 500-fold with Annexin Buffer (manufactured by Beckton Dickinson) and dispensed into three centrifuge tubes (negative control, positive control, and unstimulated sample, respectively). EDTA was added to the negative control sample and Ionomycin was added to the positive control sample, and then all samples were stained with anti-CD41 antibody (manufactured by BioLegend) and Annexin V (manufactured by Beckton Dickinson) (under light shielding, at room temperature, for 20 minutes). After staining, Annexin Buffer was added and immediately measured by FACS. Based on the obtained values of FSC (forward scatter light) and SSC (side scatter light), platelets and megakaryocytes were distinguished. And the Annexin V positive rate in iPS platelets (CD41 + fraction) in the negative control was set to 1.0 ± 0.1%, and the Annexin V negative rate of megakaryocytes in the unstimulated sample was calculated. The results are shown in Fig. 18 (where "Day 1" represents the sample before storage, "Day 5" represents the sample after 5 days of storage, and "Day 10" represents the sample after 10 days of storage). The Annexin V negative rate decreased over time in samples using either the first-generation storage solution or the second-generation storage solution. However, it was revealed that when using the second-generation storage solution, the decrease in the Annexin V negative rate was suppressed more than when using the first-generation storage solution. From these results, it was shown that samples using the second-generation storage solution contained a higher proportion of viable cells with stable cell membranes that had not undergone apoptosis. Therefore, this example demonstrated that the second-generation storage solution is also effective for the storage of iPS cell-derived megakaryocytes.

Example

[0093] [Storage of T cells] (12-1) Preparation of T cell storage solution 3% trehalose was added to lactated Ringer's solution (Lactated Ringer's Injection; manufactured by Otsuka Pharmaceutical Factory, Inc.) (hereinafter, such a solution may be referred to as "CSP-11 solution"). To the above CSP-11 solution, a VC preparation (1000 mg / L, manufactured by Sawai Pharmaceutical Co., Ltd.) and / or nicotinic acid (400 mg / L, manufactured by Toa Aiyo Co., Ltd.) were added to prepare the following four types of T cell storage solutions. CSP-11 CSP-11 + VC CSP-11 + nicotinic acid CSP-11 + VC + nicotinic acid

[0094] (12-2) Storage of T cells Commercially available frozen CD8-positive T cells (manufactured by Veritas) were thawed, washed with lymphocyte culture medium (LGM3, manufactured by Lonza), and then incubated for about 1 hour (37 °C, 5% CO 2 2). The required amount of CD8-positive T cells was aliquoted and centrifuged for 10 minutes (300 × g, room temperature). After removing the supernatant, the cells were resuspended using the TLY CULTURE KIT 25 (manufactured by GC Lymphotec) and cultured and proliferated under the conditions of 37 °C, 5% CO 2 2 (T cell concentration: approximately 1.1 × 106 cells / 5 ml). Seven days after the start of the expansion culture, the cells were washed with the CSP-11 solution, dispensed into a stem tube (manufactured by Sumitomo Bakelite Co., Ltd.), and centrifuged for 10 minutes (300×g, room temperature). After removing the supernatant, the preservation solution prepared in the above (12-1) was added and suspended (T cell concentration: about 5×10 5 cells / 1 ml). 20 μL of the cell suspension was aliquoted from each stem tube, mixed with 20 μL of trypan blue (manufactured by Gibco), and the survival rate was measured using a one-cell counter (manufactured by Biomedical Science Co., Ltd.) (the total number of cells and the number of dead cells in the area of the cell counting chambers at the four corners of one cell counting part were counted). Also, after storing the above cell suspension at 5°C for 48 hours, the survival rate was measured in the same manner.

[0095] The results are shown in Fig. 19. There was no difference in the survival rate before storage among the respective preservation solutions (Fig. 19). On the other hand, after storage for 48 hours, it became clear that the survival rate was significantly higher when using the preservation solution with VC alone or with VC and nicotinic acid added (Fig. 19). Furthermore, compared with VC alone, the preservation solution with VC and nicotinic acid added showed a significantly higher survival rate. Therefore, it was shown that the preservation solution of the present invention is also effective for refrigerated storage of T cells.

Example

[0096] [Storage of T cells] (13-1) Preparation of T cell preservation solution To the CSP-01 solution, a VC preparation (1000 mg / L, manufactured by Sawai Pharmaceutical Co., Ltd.), nicotinic acid (400 mg / L, manufactured by Toa Aiyo Co., Ltd.), and / or glucose (80 mg / dL, manufactured by Otsuka Pharmaceutical Factory, Inc.) were added to prepare the following four types of T cell preservation solutions. CSP-01 CSP-01 + VC + nicotinic acid CSP-01 + glucose CSP-01 + glucose + VC + nicotinic acid

[0097] (13-2) Storage of T cells Commercially available frozen CD8-positive T cells (manufactured by Veritas) were thawed, washed with lymphocyte culture medium (LGM3, manufactured by Lonza), and then incubated for about 6 hours (37 °C, 5% CO 2 ). The required amount of CD8-positive T cells was collected and centrifuged for 10 minutes (300×g, room temperature). After removing the supernatant, the cells were suspended using the TLY CULTURE kit 25 (manufactured by GC Lymphotec), and cultured and proliferated under the conditions of 37 °C, 5% CO 2 (T cell concentration: about 1.2×10 6 cells / 5 ml). Six days after the start of the expansion culture, the cells were washed with lactated Ringer's solution containing 3% trehalose, dispensed into stem tubes (manufactured by Sumitomo Bakelite), and centrifuged for 10 minutes (300×g, room temperature). After removing the supernatant, the preservation solution prepared in the above (12-1) was added and suspended (T cell concentration: about 5×10 5 cells / 1 ml). 20 μL of the cell suspension was collected from each stem tube, mixed with 20 μL of trypan blue (manufactured by gibco), and the viability was measured using a one-cell counter (manufactured by Biomedical Science) (the total number of cells and the number of dead cells in the areas of the cell counting chambers at the four corners of one cell counting part were counted). Also, after storing the above cell suspension at 5 °C for 24 or 48 hours, the viability was measured in the same manner. From the viability at each obtained time point, the viable cell recovery rate was calculated using the following formula 3.

[0098] [Formula 3] Viable cell recovery rate (%) = (number of viable cells after storage) ÷ (number of viable cells before storage) × 100

[0099] The results are shown in Figure 20. There was no difference in the viability before storage among the respective preservation solutions (left graph in Figure 20). On the other hand, after storage for 24 and 48 hours, it became clear that the use of the preservation solution added with VC and nicotinic acid resulted in higher viability and viable cell recovery rate (central and right graphs in Figure 20). Therefore, it was shown that the preservation solution of the present invention is also effective for refrigerated storage of T cells. Also, it became clear that in addition to VC and nicotinic acid, the addition of glucose tended to improve the viability and viable cell recovery rate.

Example

[0100] [Inhibitory effect by VB2] (14-1) Preparation of preservation solution added with water-soluble vitamin group Water-soluble vitamin group (B1, VB2, VB3, VB5, VB6, VB7, VB9, VB12, and VC) was added to the first-generation preservation solution (including 20% ACD-A solution) (hereinafter, such a preservation solution may be referred to as "first-generation + water-soluble vitamin"). Also, a group excluding VB2 from the above water-soluble vitamin group (B1, VB3, VB5, VB6, VB7, VB9, VB12, and VC) was added to the first-generation preservation solution (including 20% ACD-A solution) (hereinafter, such a preservation solution may be referred to as "first-generation + water-soluble vitamin (excluding VB2)"). Each preservation solution was adjusted to pH 7.3 ± 0.1 using 1M NaOH. The above preservation solution was added to the iPS cell-derived platelet preparation prepared by the method described in Example 1, and gently suspended to form a uniform suspension (platelet concentration: about 0.3×10 9 plts / mL). Each suspension was immediately, or seeded in a 24-well plate and horizontally shaken and stored for 5 days (under light shielding, 22 degrees, 50 rpm), and then used for the following experiments (13-2) and (13-3).

[0101] (14-2) Change in Annexin V positive rate of platelet samples By the method described in (2-3) of Example 2, the Annexin V (deterioration marker) positive rate in the platelet samples obtained in the above (14-1) was measured, and the platelet deterioration inhibitory effect of each preservation solution was examined. The results are shown in Figure 21. In the figure, "Day1" indicates the sample before preservation, and "Day5" indicates the sample after 5-day preservation. Platelets after 5-day preservation (CD41 +The Annexin V positive rate of the platelet samples) was 52.0% when the first-generation preservation solution was used, while it decreased to 43.0% in the preservation solution supplemented with the water-soluble vitamin group. Moreover, the Annexin V positive rate further decreased to 40.3% in the preservation solution supplemented with the water-soluble vitamin group (excluding VB2). These results indicated that the platelet deterioration inhibitory effects of VC and VB3 might be inhibited by VB2.

[0102] (14-3) Influence of each preservation solution on the P-Selectin positive rate of platelet samples without stimulation and the PAC-1 / P-Selectin positive rate upon ATR stimulation By the method described in (2-4) of Example 2, the P-Selectin (deterioration marker) positive rate of the platelet samples obtained in the above (14-1) without stimulation and the PAC-1 / P-Selectin (reactivity marker) positive rate upon ATR stimulation were measured to examine the platelet deterioration inhibitory effects of each preservation solution. The P-Selectin positive rate without stimulation is shown on the left side of FIG. 22, and the PAC-1 / P-Selectin positive rate upon ATR stimulation is shown on the right side of FIG. 22. The P-Selectin positive rate increased from 27.4% before preservation (Day1) to 42.9% after 5-day preservation (Day5) when the first-generation preservation solution was used. On the other hand, the P-Selectin positive rate after 5-day preservation was suppressed low to 34.9% when the preservation solution supplemented with the water-soluble vitamin group was used, and was further suppressed low to 24.0% when the preservation solution supplemented with the water-soluble vitamin group (excluding VB2) was used. Moreover, the PAC-1 / P-Selectin positive rate upon ATR stimulation decreased from 35.6% before preservation (Day1) to 32.4% after 5-day preservation (Day5) when the first-generation preservation solution was used. On the other hand, it was 36.8% even after 5-day preservation when the preservation solution supplemented with the water-soluble vitamin group was used. Furthermore, it became clear that it increased to 43.0% after 5-day preservation when the preservation solution supplemented with the water-soluble vitamin group (excluding VB2) was used. These results indicated that the platelet function maintenance effects of VC and VB3 might be inhibited by VB2.

Example

[0103] [Long-term preservation of human mesenchymal stem cells] (15-1) Preparation of mesenchymal stem cell preservation solution According to the description in (10-1) of Example 10, a CSP-01 solution was prepared. To such CSP-01 solution, a VC preparation (1000 mg / L, manufactured by Sawai Pharmaceutical Co., Ltd.) and nicotinic acid (400 mg / L, manufactured by Toa Aiyo Co., Ltd.) were added to prepare a mesenchymal stem cell preservation solution. In addition, a control preservation solution was prepared by adding distilled water (Otsuka distilled water; manufactured by Otsuka Pharmaceutical Factory, Inc.), which is a solvent, to the CSP-01 solution instead of VC and nicotinic acid.

[0104] (15-2) Preservation of mesenchymal stem cells Human bone marrow-derived mesenchymal stem cells (manufactured by Lonza) were suspended using the preservation solution prepared in (15-1) above (5x10 5 cells / mL). After the suspension was allowed to stand at 5°C for 1, 2, 4, 7, 14, 21, 28, 35, and 63 days, the total cell number and the number of dead cells were measured using a microscope. The cell viability (%) and viable cell recovery rate (%) at each time point were calculated using Formulas 1 and 2 described in (10-2) of Example 10.

[0105] The results are shown in Figure 23. When the control preservation solution (labeled as “+ solvent” in the figure) was used, it was revealed that the cell viability and viable cell recovery rate decreased rapidly after 2 days of preservation. On the other hand, when the preservation solution added with VC and nicotinic acid (labeled as “+VC+nicotinic acid” in the figure) was used, a rapid decrease in the cell viability and viable cell recovery rate was not observed, and it was revealed that they were maintained at approximately the same level as before preservation even after 35 days. Also, when the preservation solution added with VC and nicotinic acid was used, it was shown that the cell viability and viable cell recovery rate were significantly higher even after 63 days of preservation compared to the control preservation solution. From these results, it was shown that VC and nicotinic acid exhibit excellent effects even in the long-term preservation of mesenchymal stem cells.

Example

[0106] [Long-term preservation of human mesenchymal stem cells] (16-1) Preparation of mesenchymal stem cell preservation solution To CSP-01 solution or lactated Ringer's solution (Lactec infusion; manufactured by Otsuka Pharmaceutical Factory, Inc.), a VC preparation (1000 mg / L, manufactured by Sawai Pharmaceutical Co., Ltd.) and nicotinic acid (400 mg / L, manufactured by Toa Aiyo Co., Ltd.) were added to prepare the following four kinds of mesenchymal stem cell preservation solutions. CSP-01 CSP-01 + VC + nicotinic acid Lactated Ringer's solution Lactated Ringer's solution + VC + nicotinic acid

[0107] (16-2) Preservation of mesenchymal stem cells Using the preservation solution prepared according to the above (16-1), human adipose-derived mesenchymal stem cells (manufactured by Lonza) were suspended (5x10 5 cells / mL). After the suspension was allowed to stand at 5°C for 7, 14, 21, and 28 days, the total cell number and the number of dead cells were measured using a microscope. The cell viability (%) and viable cell recovery rate (%) at each time point were calculated using Formulas 1 and 2 described in (10-2) of Example 10.

[0108] The results are shown in Fig. 24. When only CSP-01 or lactated Ringer's solution was used, the cell viability and viable cell recovery rate significantly decreased at the time point after 7 days of preservation. On the other hand, when the preservation solution added with VC and nicotinic acid was used, it was revealed that the decrease in cell viability and viable cell recovery rate was suppressed. In particular, when CSP-01 + VC + nicotinic acid was used, it was revealed that both the cell viability and viable cell recovery rate were maintained at a high level even after 28 days of preservation. Also, when lactated Ringer's solution + VC + nicotinic acid was used, it was revealed that both the cell viability and viable cell recovery rate were maintained at a high level even after 14 days of preservation. From these results, it was shown that VC and nicotinic acid have excellent effects even in the long-term preservation of human adipose-derived mesenchymal stem cells.

Example

[0109] [Long-term preservation of human mesenchymal stem cells] (17-1) Preparation of mesenchymal stem cell preservation solution To the CSP-01 solution, a VC preparation (1000 mg / L, manufactured by Sawai Pharmaceutical Co., Ltd.) and / or nicotinic acid (400 mg / L, manufactured by Toa Aiyo Co., Ltd.) were added, and further a sodium bicarbonate preparation (Mayron Intravenous 8.4%, manufactured by Otsuka Pharmaceutical Factory, Inc.) was added to adjust the pH to 7.0 - 7.3. Also, as a control preservation solution, to the CSP-01 solution, a sodium bicarbonate preparation (Mayron Intravenous 8.4%, manufactured by Otsuka Pharmaceutical Factory, Inc.) was added to adjust the pH to 7.0 - 7.3. In this way, the following 4 types of mesenchymal stem cell preservation solutions were prepared. CSP-01 CSP-01 + VC CSP-01 + nicotinic acid CSP-01 + VC + nicotinic acid

[0110] (17 - 2) Preservation of mesenchymal stem cells Using the preservation solution prepared in the above (17 - 1), human adipose-derived mesenchymal stem cells (manufactured by Lonza) were suspended (5x10 5 cells / mL). After the suspension was allowed to stand at 5°C for 7, 14, 21, and 28 days, the total cell number and the number of dead cells were measured using a microscope. The cell viability (%) and viable cell recovery rate (%) at each time point were calculated using Formulas 1 and 2 described in (10 - 2) of Example 10.

[0111] The results are shown in Figure 25. When CSP-01 + nicotinic acid was used, the cell viability and viable cell recovery rate decreased linearly in the same manner as the control (CSP-01). On the other hand, when CSP-01 + VC was used, the decrease in cell viability and viable cell recovery rate was significantly suppressed. Furthermore, when CSP-01 + VC + nicotinic acid was used, the cell viability and viable cell recovery rate were more significantly improved. From these results, in the long-term preservation of mesenchymal stem cells, VC is effective alone, but it was shown that a more excellent effect is exerted by the combined use of VC and nicotinic acid.

Example

[0112] [Preservation of mesenchymal stem cells from young pig bone marrow] (18 - 1) Preparation of mesenchymal stem cell preservation solution To the CSP-01 solution, a VC preparation (1000 mg / L, manufactured by Sawai Pharmaceutical Co., Ltd.) and nicotinic acid (400 mg / L, manufactured by Toa Aiyo Co., Ltd.) were added to prepare a mesenchymal stem cell preservation solution (CSP-01 + VC + nicotinic acid). Also, as a control preservation solution, only the CSP-01 solution was used.

[0113] (18-2) Preservation of Mesenchymal Stem Cells According to the method of Nishimura et al. (Xenotransplantation. 2019 May;26(3):e12501.), mesenchymal stem cells derived from juvenile pig bone marrow (np mesenchymal stem cells) were prepared. Such np mesenchymal stem cells were suspended using the preservation solution prepared in (18-1) above (5x10 5 cells / mL). After the suspension was allowed to stand at 5°C for 7, 14, 21, and 28 days, the total cell number and the number of dead cells were measured using a microscope. The cell viability (%) and viable cell recovery rate (%) at each time point were calculated using Formulas 1 and 2 described in (10-2) of Example 10.

[0114] The results are shown in Figure 26. When CSP-01 + VC + nicotinic acid was used, the viability and viable cell recovery rate of np mesenchymal stem cells were improved at all preservation periods compared to the control (CSP-01). Also, it became clear that the viability and viable cell recovery rate of np mesenchymal stem cells were maintained at a high level even after 28 days of preservation with CSP-01 + VC + nicotinic acid. From these results, it was shown that the preservation solution added with VC and nicotinic acid also has an excellent effect in the preservation of np mesenchymal stem cells.

Example

[0115] [Preservation of T Cells] (19-1) Preparation of T Cell Preservation Solution To the lactated Ringer's solution (Lacteck Infusion; manufactured by Otsuka Pharmaceutical Factory, Inc.), a VC preparation (1000 mg / L, manufactured by Sawai Pharmaceutical Co., Ltd.), nicotinic acid (400 mg / L, manufactured by Toa Aiyo Co., Ltd.), and / or glucose (80 mg / dL, manufactured by Otsuka Pharmaceutical Factory, Inc.) were added to prepare the following 8 types of T cell preservation solutions. LR LR + Glucose LR + VC LR + Nicotinic acid LR + VC + Nicotinic acid LR + VC + Glucose LR + Nicotinic acid + Glucose LR + VC + Nicotinic acid + Glucose

[0116] (19 - 2) Preservation of T cells Commercially available frozen CD8 - positive T cells (manufactured by Veritas) were thawed, washed with lymphocyte culture medium (LGM3, manufactured by Lonza), and then incubated for 1 hour (37°C, 5% CO 2 2). The required amount of CD8 - positive T cells was aliquoted and centrifuged for 10 minutes (300×g, room temperature). After removing the supernatant, the cells were resuspended using TLY CULTURE KIT 25 (manufactured by GC Lymphotec) and cultured and proliferated under the conditions of 37°C, 5% CO 2 2 (T - cell concentration: approximately 8×10 5 cells / 5ml). Seven days after the start of the expansion culture, the cells were washed with PBS(-), dispensed into stem tubes (manufactured by Sumitomo Bakelite), and centrifuged for 10 minutes (300×g, room temperature). After removing the supernatant, the preservation solution prepared in (19 - 1) above was added and suspended (T - cell concentration: approximately 5×10 5 cells / 1ml). At the time when the above cell suspension was stored at 5°C for 24 hours and then further stored at 25°C for 6 hours (after a total storage of 30 hours), the total cell number and the number of dead cells were measured using a microscope. The cell viability (%) and viable cell recovery rate (%) at each time point were calculated using Formulas 1 and 2 described in (10 - 2) of Example 10.

[0117] The survival rate of T cells after storage is shown in Fig. 27. As shown in the upper graph of Fig. 27, the survival rate after storage at 5°C for 24 hours was significantly increased when using VC-added storage solutions (LR+VC, LR+VC+nicotinic acid, LR+VC+glucose, and LR+VC+nicotinic acid+glucose) compared with the LR storage solution. Also, as shown in the lower graph of Fig. 27, the survival rate after storage at 5°C for 24 hours + 25°C for 6 hours was significantly increased when using VC- and glucose-added storage solutions (LR+VC+glucose, and LR+VC+nicotinic acid+glucose) compared with the LR storage solution, and furthermore, an increasing trend was observed when using the VC- and nicotinic acid-added storage solution (LR+VC+nicotinic acid).

[0118] The viable cell recovery rate of T cells after storage is shown in Fig. 28. As shown in the upper graph of Fig. 28, the viable cell recovery rate after storage at 5°C for 24 hours was significantly increased when using VC-added storage solutions (LR+VC, LR+VC+nicotinic acid, LR+VC+glucose, and LR+VC+nicotinic acid+glucose) compared with the LR storage solution. Among them, a more remarkable viable cell recovery rate improvement effect was observed in the storage solutions added with a combination of VC and glucose (LR+VC+glucose, and LR+VC+nicotinic acid+glucose). Also, as shown in the lower graph of Fig. 28, the viable cell recovery rate after storage at 5°C for 24 hours + 25°C for 6 hours was significantly increased when using VC- and glucose-added storage solutions (LR+VC+glucose, and LR+VC+nicotinic acid+glucose) compared with the LR storage solution, and furthermore, an increasing trend was observed when using the VC- and nicotinic acid-added storage solution (LR+VC+nicotinic acid).

[0119] From the above results, under the conditions assuming actual T cell transplantation, that is, after storing T cells at 5°C and then changing the temperature to 25°C for further storage, it was shown that the use of a combination of VC and nicotinic acid improved both the survival rate and the viable cell recovery rate compared with using each alone. Also, it was clarified that the use of a combination of VC and glucose further improved the survival rate and the viable cell recovery rate. [Industrial Applicability]

[0120] According to the present invention, since it is possible to perform shaking preservation for at least 10 days while maintaining the function of platelets, it is useful for the preparation of platelet preparations for disease and wound treatment. Further, according to the present invention, long-term preservation is also possible in non-freezing preservation of mesenchymal stem cells, megakaryocytes, T cells, etc., so it is useful in the field of transplantation medicine and cancer treatment in the field of regenerative medicine and the like.

Claims

1. A mixed solution comprising a storage solution and platelets, a storage solution containing 10 to 5000 mg / L of niacin or a salt thereof and 10 to 8000 mg / L of ascorbic acid or a salt thereof, the storage solution having an osmotic pressure isotonic with the platelets; and platelets stored in said storage solution, The platelets are 1.0×10 ９ When the blood is filled in a blood storage bag together with the storage solution at a platelet concentration of 100 plts / mL and stored in a light-shielded condition at 22° C. with horizontal shaking at 50 rpm for 10 days, the storage solution remains neutral. The mixture.

2. Platelets preserved in a preservation solution having the following characteristics i) and ii): i) containing 10 to 5000 mg / L of niacin or a salt thereof and 10 to 8000 mg / L of ascorbic acid or a salt thereof, and having an osmotic pressure isotonic with the platelets; ii) Add 1.0 x 10 platelets to a blood storage bag. ９ When the storage solution contains a platelet concentration of 100 plts / mL and is stored with horizontal shaking at 50 rpm at 22° C. for 10 days in the dark, the storage solution remains neutral. The platelets.

3. A platelet preparation preserved in a preservation solution having the following characteristics i) and ii): i) containing 10 to 5000 mg / L of niacin or a salt thereof and 10 to 8000 mg / L of ascorbic acid or a salt thereof, and having an osmotic pressure isotonic with the platelets; ii) Add 1.0 x 10 platelets to a blood storage bag. ９ When the storage solution contains a platelet concentration of 100 plts / mL and is stored with horizontal shaking at 50 rpm at 22° C. for 10 days in the dark, the storage solution remains neutral. The platelet preparation.

4. A storage solution for storing platelets, comprising: Contains 10 to 5000 mg / L of niacin or a salt thereof, and 10 to 8000 mg / L of ascorbic acid or a salt thereof; the osmotic pressure is isotonic with said platelets; The platelets are 1.0×10 ９ When the blood is filled in a blood storage bag together with the storage solution at a platelet concentration of 100 plts / mL and stored in a light-shielded condition at 22° C. with horizontal shaking at 50 rpm for 10 days, the storage solution remains neutral. The preservative solution.

5. A mixed solution comprising a storage solution and platelets or megakaryocytes, a storage solution for storing platelets or megakaryocytes at 20 to 24° C. for 1 to 15 days, the storage solution containing 10 to 5000 mg / L of niacin or a salt thereof and 10 to 8000 mg / L of ascorbic acid or a salt thereof in an isotonic solution and not containing vitamin B2 or a salt thereof; and platelets or megakaryocytes stored in the storage solution. The mixture.

6. Platelets preserved in a preservation solution, The preservation solution does not contain 10 to 5000 mg / L of niacin or a salt thereof, and 10 to 8000 mg / L of ascorbic acid or a salt thereof, or vitamin B2 or a salt thereof, and is for preserving platelets at 20 to 24° C. for 1 to 15 days. The platelets.

7. A platelet preparation preserved in a preservation solution, The preservation solution contains 10 to 5000 mg / L of niacin or a salt thereof, and 10 to 8000 mg / L of ascorbic acid or a salt thereof in an isotonic solution, and does not contain vitamin B2 or a salt thereof, and is a preservation solution for preserving platelets at 20 to 24° C. for 1 to 15 days. The platelet preparation.

8. 1. A method for preserving platelets, comprising: storing platelets in a storage solution containing 10 to 5000 mg / L of niacin or a salt thereof and 10 to 8000 mg / L of ascorbic acid or a salt thereof, the storage solution having an osmotic pressure isotonic with the platelets; The storage solution contains 1.0×10 ９ When the storage solution is filled in a blood storage bag with a platelet concentration of 100 plts / mL and stored at 22°C for 10 days with horizontal shaking at 50 rpm in the dark, the storage solution remains neutral. The method for preserving platelets.

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