Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/52—Chemical aspects of preservation of animal cells or human cells
  • C12N5/522—Preservation media
  • C12N5/526—Physiologically active agents, e.g. antioxidants or nutrients
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
  • A01N1/10—Preservation of living parts
  • A01N1/12—Chemical aspects of preservation
  • A01N1/122—Preservation or perfusion media
  • A01N1/126—Physiologically active agents, e.g. antioxidants or nutrients
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/14—Blood; Artificial blood
  • A61K35/19—Platelets; Megacaryocytes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/56—Physical preservation processes for animal cells or human cells
  • C12N5/562—Temperature processes, e.g. following predefined temperature changes over time
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
  • A01N1/10—Preservation of living parts
  • A01N1/12—Chemical aspects of preservation
  • A01N1/122—Preservation or perfusion media
  • A01N1/125—Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
  • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
  • A61M1/02—Blood transfusion apparatus
  • A61M1/0272—Apparatus for treatment of blood or blood constituents prior to or for conservation, e.g. freezing, drying or centrifuging
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
  • A61M2202/00—Special media to be introduced, removed or treated
  • A61M2202/04—Liquids
  • A61M2202/0413—Blood
  • A61M2202/0427—Platelets; Thrombocytes

Definitions

• the present invention relates to a novel method for preserving platelets, a platelet preservation solution, and the like. More specifically, the present invention relates to a method of preserving platelets in a refrigerated or frozen state while retaining more of their functions, and a platelet preservation solution for use in such a method.
• PRP platelet rich plasma
• EGF Epidermal growth Factor
• PDGF Platelet-Derived Growth Factor
• VEGF Vascular Endothelial Growth Factor
• IGF Insulin-like Growth Factor
• cytokines such as Insulin-like Growth Factor
• FGF Fibroblast Growth Factor
• HGF Hepatocyte Growth Factor
• TGF Transforming Growth Factor
• PRP is used in the treatment of many diseases such as knee osteoarthritis and intractable skin ulcers (for example, Patent Document 1) and in cosmetic surgery.
• PRP therapy PRP is injected into the affected area. Platelets injected into the affected area produce cytokines in the affected area, and these cytokines promote cell regeneration in the affected area and are said to play an effective role in wound healing and tissue regeneration. Therefore, in PRP therapy, it is extremely important that the cytokine production function of the platelets injected into the affected area is maintained without fail.
• Patent Document 2 there is a problem that the function of platelets deteriorates over time after blood collection
• PRP in PRP therapy, PRP is generally prepared from blood taken from the patient and then injected into the patient within several hours.If PRP therapy is performed multiple times, each time It is common to prepare PRP by drawing blood from a patient. In this way, in PRP therapy, it is extremely important that the cytokine production function of the platelets injected into the affected area is maintained, so there is no concept of preserving PRP for more than a few hours. There was no thought of storing it in a refrigerator or freezing it.
• platelet preparations for blood transfusions are stored for about four days at room temperature from the date of manufacture. From the viewpoint of maintaining platelet functions such as platelet aggregation ability, room temperature is considered to be essential as the storage temperature.
• An object of the present invention is to provide a method for preserving platelets in a refrigerated or frozen state while retaining more of their functions, and a platelet preservation solution for use in such a method.
• the present inventors suspended platelets in a platelet preservation solution containing an electrolyte and an anticoagulant, and stored the suspension refrigerated or frozen at a temperature below 10°C.
• the present inventors have discovered that platelets can be preserved while preserving their functions by preserving them, leading to the completion of the present invention.
• the present invention (1) Platelet preservation solution for refrigeration or freezing, containing electrolytes and anticoagulants; (2)
• the electrolyte is one or more selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium hydrogen carbonate, sodium lactate, and sodium acetate, and is an anticoagulant.
• the platelet preservation solution according to (1) above which is one or more selected from the group consisting of salts thereof; (3) A platelet-containing composition comprising the platelet preservation solution described in (1) or (2) above and platelets; (4) A method for producing a platelet-containing composition, comprising the step of suspending platelets in the platelet preservation solution described in (1) or (2) above; (5) A method for preserving platelets, comprising the steps of suspending platelets in the platelet preservation solution described in (1) or (2) above, and storing the suspension refrigerated or frozen at 10° C.
• the step of suspending the platelets in the platelet preservation solution is a step of suspending the platelets in the platelet preservation solution in a volume that is 0.1 times or more larger than the volume of plasma in the platelets.
• the present invention it is possible to provide a method for preserving platelets in a refrigerated or frozen state while retaining more of their functions, and a platelet preservation solution for use in such a method.
• FIG. 1 is a diagram showing the results of comparing the cytokine secretion ability of platelets.
• the vertical axis represents the concentration (pg/mL) of VEGF (vascular endothelial growth factor) in the platelet culture solution.
• VEGF vascular endothelial growth factor
• FIG. 2 is a diagram showing the results of comparing the aggregation ability of platelets.
• the vertical axis represents platelet aggregation rate (%).
• the bar graph on the left in the figure represents the results when stored at -20°C
• the bar graph on the right represents the results when stored at 4°C.
• FIG. 3 is a diagram showing the results of comparing the cytokine secretion ability per platelet cell piece. The vertical axis is expressed as a ratio to the VEGF concentration when stimulated with CaCl 2 after storage under storage condition 1 as a standard.
• FIG. 4 is a diagram showing the results of comparing platelet aggregation ability. The vertical axis represents platelet aggregation rate (%). This result indicates that the adhesion function of platelet membrane receptors is maintained and the signal transduction ability of platelet aggregation via membrane receptors is maintained.
• FIG. 3 is a diagram showing the results of comparing the cytokine secretion ability per platelet cell piece. The vertical axis is expressed as a ratio to the VEGF concentration when stimulated with CaCl 2 after storage under storage condition 1 as a standard.
• FIG. 4 is a diagram showing the results of comparing platelet aggregation ability. The vertical axis represents platelet aggregation rate (%). This result indicates that the adhesion function
• FIG. 5 is a diagram showing the results of comparing platelet concentration concentration ratios.
• the vertical axis represents the ratio of the platelet concentration in the sample after concentration, when the platelet concentration in the sample before concentration is 1.
• the time on the horizontal axis represents the storage time of donor blood used for sample preparation.
• FIG. 6 is a diagram showing the results of comparing the aggregation ability of platelets.
• the vertical axis represents the maximum platelet aggregation rate (%).
• the time on the horizontal axis represents the storage time of donor blood used for sample preparation.
• FIG. 7 is a diagram showing the results of comparing platelet concentration concentration ratios.
• the vertical axis represents the ratio of the platelet concentration in the sample after concentration, when the platelet concentration in the sample before concentration is 1.
• FIG. 8 is a diagram showing the results of comparing platelet concentrations before and after preservation with the platelet preservation solution of the present invention.
• the vertical axis represents platelet concentration ( ⁇ 10 3 cells/ ⁇ L).
• the bar graph on the left represents the platelet concentration of the initial PRP before storage for 3 weeks
• the bar graph on the right represents the platelet concentration of the initial PRP after storage for 3 weeks.
• FIG. 9 represents the ratio (“maximum aggregation change rate”) (%) of the maximum aggregation ability of platelets after storage to the maximum aggregation ability of platelets before storage.
• the vertical axis represents the ratio of the platelet concentration in the sample after concentration, when the platelet concentration in the sample before concentration is 1.
• #1 and #2 on the horizontal axis represent donor numbers assigned to identify each donor.
• FIG. 10 is a diagram showing the results of comparing the VEGF secretion ability of platelets.
• the VEGF secretion ability on the vertical axis is expressed as a relative value when the VEGF secretion ability of PRP before storage prepared from blood donor #1 is taken as 100.
• #1 and #2 on the horizontal axis represent donor numbers assigned to identify each donor.
• the bar graph on the left represents the VEGF secretion ability of PRP before storage for 3 weeks
• the bar graph on the right represents the VEGF secretion ability of PRP after storage for 3 weeks.
• the present invention [1] A platelet preservation solution for refrigeration or freezing containing an electrolyte and an anticoagulant (hereinafter also referred to as “platelet preservation solution of the present invention”); [2] A platelet-containing composition containing the platelet preservation solution of the present invention and platelets (hereinafter also referred to as “platelet-containing composition of the present invention”); [3] Step of suspending platelets in the platelet preservation solution of the present invention, method for producing a platelet-containing composition (hereinafter also referred to as “the production method of the present invention”) [4] Suspending platelets in the platelet preservation solution of the present invention, and A method for preserving platelets (hereinafter also referred to as "preservation method of the present invention”), which includes a step of storing a suspension in a refrigerator or frozen at 10° C. or lower; It includes embodiments such as.
• platelets include mammalian platelets, more specifically humans, mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, horses, cows, sheep, pigs, goats, Examples include platelets from monkeys and the like, and human platelets are particularly preferred.
• the platelets in this specification may be platelets for blood transfusion, but from the viewpoint of enjoying the significance of the present invention to a greater extent, platelets for promoting tissue repair such as for PRP therapy are preferably used.
• the method for preparing platelets is not particularly limited, and platelets may be isolated or concentrated from mammalian blood, or platelets obtained by inducing differentiation of mammalian pluripotent stem cells, etc. It may be.
• platelets isolated or concentrated from mammalian blood include platelet-rich plasma (PRP) and washed platelets, and platelet-rich plasma is preferred from the viewpoint of ease of preparation.
• the platelets do not have to be platelets originating from the subject to be administered, but are preferably platelets originating from the subject from the viewpoint of lowering the possibility of rejection.
• the method for preparing platelets from the blood of mammals such as humans is not particularly limited, and any known method can be used. Specifically, a method for obtaining platelet-rich plasma by centrifuging blood may be mentioned. The centrifugation process may be carried out once (One step centrifugation) or twice (Two step centrifugation). One-step centrifugation includes, for example, centrifugation at 100-300G for 5-20 minutes, and two-step centrifugation includes, for example, centrifugation at 100-300G for 5-20 minutes, followed by further centrifugation at 200-300G. Examples include a method of centrifugation for 5 to 20 minutes. Note that when platelets are prepared from blood, the blood may be collected from a mammal and then stored.
• Examples of the storage period include within 120 hours, within 96 hours, within 72 hours, and within 48 hours.
• Storage temperature is not particularly limited, whether refrigerated or frozen, as long as it is below 10°C, but from the perspective of preserving more platelet function, it should be higher than 0°C and below 5°C. , or 0°C or lower.
• Examples of the temperature range of 0°C or lower include -80°C or higher and 0°C or lower, -60°C or higher and -5°C or lower, -40°C or higher and -10°C or lower, or -30°C or higher and -15°C or lower.
• the platelet preservation solution of the present invention is not particularly limited as long as it contains an electrolyte and an anticoagulant.
• electrolyte is not particularly limited as long as an aqueous solution containing the electrolyte can be administered to any mammal, and includes, for example, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium hydrogen carbonate, One or more selected from the group consisting of sodium lactate and sodium acetate.
• the concentration of electrolytes contained in the platelet preservation solution of the present invention is not particularly limited as long as the effects of the present invention can be obtained, but the concentration is, for example, 160 to 400 mEq/L, preferably 160 to 400 mEq/L as the total amount of electrolytes (mEq/L). is 180 to 360 mEq/L, more preferably 180 to 320 mEq/L.
• a powdered electrolyte When preparing the platelet preservation solution of the present invention, a powdered electrolyte may be used, or a commercially available electrolyte infusion solution may be used.
• electrolyte infusions include physiological saline, Ringer's solution, lactated Ringer's solution (including sugar-added lactated Ringer's solution), acetic acid Ringer's solution (including sugar-added acetate Ringer's solution), bicarbonate Ringer's solution (including sugar-added ones), etc.
• Ringer's bicarbonate solution is preferred. These may be used alone or in combination of two or more.
• anticoagulant include chelating agents, which suppress blood coagulation by coordinating calcium ions, which are a factor in blood coagulation, and antithrombin drugs, including heparin, which suppress thrombin activity, which is a factor in blood coagulation.
• chelating agents are preferred.
• Anticoagulants that are chelating agents include citric acid, EDTA (ethylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), DCTA (1,2-diaminocyclohexanetetraacetic acid), and EGTA (ethylene glycol bis-2-aminoethyl ether tetraacetic acid).
• aqueous solutions containing anticoagulants include ACD-A solution (aqueous solution containing sodium citrate hydrate, citric acid hydrate, and glucose), CPD solution (sodium citrate hydrate), aqueous solution containing citric acid hydrate, glucose and sodium dihydrogen phosphate hydrate), CPDA-1 solution (sodium citrate hydrate, citric acid hydrate, glucose, sodium dihydrogen phosphate hydrate)
• ACD-A solution is particularly preferred.
• the anticoagulant only one type may be used, or two or more types may be used in combination. Commercially available anticoagulants and anticoagulants can be used.
• the concentration of the anticoagulant in the preservation solution is not particularly limited as long as the effects of the present invention can be obtained, but for example, when the anticoagulant is citric acid or a salt thereof, the concentration of the anticoagulant in the platelet preservation solution of the present invention is For example, the concentration in terms of citric acid is 0.2 to 3 w/v%, preferably 0.5 to 2 w/v%. Similarly, when the anticoagulant is EDTA or a salt thereof, the concentration in terms of EDTA is, for example, 0.2 to 3 w/v%, preferably 0.5 to 2 w/v%, and the anticoagulant is DTPA or a salt thereof.
• the concentration is 0.2 to 3 w/v%, preferably 0.5 to 2 w/v% in terms of DTPA, and when the anticoagulant is DCTA or a salt thereof, the concentration is calculated as DCTA.
• the concentration is, for example, 0.2 to 3 w/v%, preferably 0.5 to 2 w/v%, and when the anticoagulant is EGTA or a salt thereof, the concentration in terms of EGTA is, for example, 0.2 to 3 w/v%.
• the concentration in terms of oxalic acid is, for example, 0.2 to 3 w/v%, preferably 0. Examples include 5 to 2 w/v%.
• the total concentration can be, for example, 0.2 to 3 w/v%, preferably 0.5 to 2 w/v%.
• a powdered anticoagulant or a commercially available anticoagulant aqueous solution containing an anticoagulant or the like may be used.
• the method for preparing the platelet preservation solution of the present invention is not particularly limited, and may be prepared by mixing an electrolyte, an anticoagulant, and water, or may be prepared by mixing an electrolyte infusion and an anticoagulant.
• the volume ratio of the electrolyte infusion to the anticoagulant is, for example, 0.5:1 to 10:1, preferably 1:1 to 5: 1, more preferably 1.5:1 to 3.5:1, still more preferably 1.8:1 to 3:1.
• the concentration of the electrolyte infusion in the storage solution is, for example, 20 to 90 v/v%, preferably 40 to 90 v/v%, more preferably 60 to 80 v/v%, More preferably, it is 65 to 75 v/v%.
• the osmotic pressure of the platelet preservation solution of the present invention is not particularly limited as long as the effects of the present invention can be obtained, but for example, 230 to 400 mOsm/L, preferably 250 to 380 mOsm/L, more preferably 265 to 350 mOsm/L. , more preferably 270 to 330 mOsm/L, more preferably 275 to 310 mOsm/L, even more preferably 280 to 300 mOsm/L.
• the platelet preservation solution of the present invention may contain only an electrolyte, an anticoagulant, and water, but may also contain other optional components.
• Such optional ingredients include sugars such as glucose and maltose.
• the concentration of sugar contained in the platelet preservation solution of the present invention is not particularly limited as long as the effects of the present invention can be obtained, but the total concentration of sugar in the platelet preservation solution is, for example, 0.1 to 25 w/v%, Alternatively, examples include 0.5 to 20 w/v%, 1 to 10 w/v%, or 2 to 5 w/v%.
• the platelet preservation solution of the present invention may be housed in a container.
• Such containers can include bags, syringes, ampoules, vials, and the like.
• the method for preserving platelets of the present invention includes: suspending platelets in the platelet preservation solution of the present invention, and There are no particular limitations as long as the process includes a step of storing the suspension under refrigeration or freezing at 10°C or lower.
• step of suspending platelets in the platelet preservation solution of the present invention is a step of suspending platelets in the platelet preservation solution of the present invention.
• suspension step is a step of suspending platelets in the platelet preservation solution of the present invention.
• platelets may be suspended by adding platelet preservation solution, platelet preservation solution may be added to platelets and suspended, or electrolyte infusion may be added to platelets and then an anticoagulant may be added.
• the platelets may be suspended, or the platelets may be suspended by adding electrolyte infusion and then an anticoagulant, or the electrolyte infusion and anticoagulant may be added to platelets and suspended, or the platelets may be suspended by adding electrolyte infusion and anticoagulant.
• a coagulant may be added to platelets and then an electrolyte infusion may be added and suspended, or platelets may be added to an anticoagulant and then an electrolyte infusion is added and suspended.
• the amount of the platelet preservation solution of the present invention used in the suspension step is not particularly limited as long as the effects of the present invention can be obtained, but for example, the amount of the platelet preservation solution of the present invention is 0.1 times or more the volume of plasma in platelets.
• a platelet preservation solution is mentioned, and from the viewpoint of retaining more platelet functions, it is preferably 0.3 times or more, more preferably 0.5 times or more, still more preferably 0.8 times or more, and even more preferably 1 time or more.
• a volume of platelet preservation solution may be mentioned.
• the upper limit of the amount of the platelet preservation solution of the present invention to be used is not particularly limited, and may be, for example, 1000 times or less, 500 times or less, 200 times or less relative to the volume of plasma in platelets.
• step of refrigerated or frozen preservation of the suspension at 10°C or less involves the refrigerated or frozen preservation of the suspension obtained in the suspension step. There is no particular restriction as long as it is a process.
• the storage temperature in the storage process is not particularly limited, whether it is refrigerated storage or frozen storage, as long as it is 10°C or lower, but from the viewpoint of retaining more platelet function, it is higher than 0°C. C. or lower or 0.degree. C. or lower is preferred.
• Examples of the temperature range of 0°C or lower include -80°C or higher and 0°C or lower, -60°C or higher and -5°C or lower, -40°C or higher and -10°C or lower, or -30°C or higher and -15°C or lower.
• the storage period of the storage step is not particularly limited, but from the viewpoint of enjoying the significance of the present invention more, for example, 3 hours or more, preferably 1 day or more, more preferably 3 days or more, even more preferably 7 days or more, More preferably, the period is 10 days or more, 15 days or more, or 25 days or more. Further, the upper limit of the storage period includes, for example, 6 months or less, 3 months or less, 2 months or less, or 1 month or less.
• the method for producing the platelet-containing composition of the present invention is not particularly limited as long as it includes the step of suspending platelets (preferably PRP) in the platelet preservation solution of the present invention.
• the suspension step is as described above in the method for preserving platelets of the present invention.
• the method for producing a platelet-containing composition of the present invention preferably further includes a step of preparing platelets (preferably PRP) before the suspending step.
• methods for preparing platelets include methods of isolating or concentrating platelets from mammalian blood, and methods of inducing differentiation of mammalian pluripotent stem cells into platelets. From this point of view, a method of isolating or concentrating platelets from mammalian blood is preferred. Examples of methods for isolating or concentrating platelets from mammalian blood include a method of centrifuging blood. The centrifugation process may be carried out once (One step centrifugation) or twice (Two step centrifugation).
• One-step centrifugation includes, for example, centrifugation at 100-300G for 5-20 minutes, and two-step centrifugation includes, for example, centrifugation at 100-300G for 5-20 minutes, followed by 5-20 minutes at 200-300G.
• An example is a method of centrifugation for 20 minutes.
• the platelet-containing composition of the present invention is not particularly limited as long as it contains the platelet preservation solution of the present invention and platelets.
• the platelet-containing composition of the present invention may be a platelet preparation.
• the concentration of platelets in the platelet-containing composition of the present invention is not particularly limited, but for example, 1 ⁇ 10 3 to 1 ⁇ 10 30 cells/mL, preferably 1 ⁇ 10 5 to 1 ⁇ 10 25 cells/mL, more preferably Examples include 1 ⁇ 10 7 to 1 ⁇ 10 20 cells/mL.
• the platelet-containing composition of the present invention may contain only the platelet preservation solution of the present invention and platelets, but may also contain other optional components.
• the platelet-containing composition of the present invention can be produced by mixing platelets and the platelet preservation solution of the present invention.
• retaining more platelet functions means that the platelet preservation solution of the present invention retains more of the platelet function than when the same platelets are stored under the same storage conditions without using the platelet preservation solution of the present invention. This means that platelets retain more of their function when preserved using Moreover, in this specification, “platelet function” means cytokine secretion ability and/or platelet aggregation ability. As the cytokine secretion ability, VEGF secretion ability is preferably mentioned.
• Ringer's bicarbonate solution (“Bicarbon”) is shown in Table 1, and the composition of ACD-A solution is shown in Table 2.
• the above ACD-A solution contains 2.2 w / v% sodium citrate hydrate (C 6 H 5 Na 3 O 7.2H 2 O) and 0.8 w/v% citric acid hydrate.
• the above Ringer 's bicarbonate solution contains 0.049 w/ v % sodium citrate hydrate (C 6 H 5 Na 3 O 7 . 2H 2 O). Therefore, the citric acid concentration (citric acid equivalent concentration) in the platelet preservation solution of the present invention obtained by mixing Ringer's bicarbonate solution and ACD-A solution at a volume ratio of 2.3:1 is approximately 0. It is .75 w/v%.
• the electrolyte of the bicarbonate Ringer's solution is 286 mEq/L
• the electrolyte of the ACD-A solution is 57 mEq/L. Therefore, the electrolyte of the platelet preservation solution of the present invention obtained by mixing Ringer's bicarbonate solution and ACD-A solution at a volume ratio of 2.3:1 is about 217 mEq/L.
• Storage conditions 1 PRP only
• PRP was placed in a syringe and sealed, and then stored at 4°C or -20°C for 10 or 17 days.
• Test 3 Effects of long-term storage of blood samples In Tests 1 and 2, PRP prepared from blood immediately collected from a donor was used. Therefore, the following test was conducted using PRP prepared from blood collected from a donor and stored for a long time.
• the platelet preservation solution of the present invention was produced by the method described in Test 2 above.
• the results of evaluating the platelet concentration concentration rate in the first PRP are shown in FIG. As can be seen from FIG. 5, the platelet concentration concentration rate of the first PRP was maintained at approximately the same level even when donor blood (whole blood) was stored for 120 hours and when it was stored for 48 hours.
• the platelet aggregation ability of the initial PRP was evaluated using the same method as described in Test 1 above. The results are shown in FIG. As can be seen from FIG. 6, the maximum agglutination rate of the initial PRP was maintained at approximately the same level even when donor blood (whole blood) was stored for 120 hours and when stored for 48 hours.
• the platelet preservation solution of the present invention was produced by the method described in Test 2 above.
• FIG. 7 shows the results of evaluating the platelet concentration concentration rate in the first PRP and the platelet concentration concentration rate after the first PRP was stored for 3 weeks using the platelet preservation solution of the present invention. As can be seen from FIG. 7, in all samples, the platelet concentration concentration rate of PRP preserved with the platelet preservation solution of the present invention was maintained at approximately the same level as the platelet concentration concentration rate of initial PRP.
• the present invention it is possible to provide a method for preserving platelets in a refrigerated or frozen state while retaining more of their functions, and a platelet preservation solution for use in such a method.

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• 2023
  • 2023-05-15 JP JP2024521916A patent/JPWO2023223984A1/ja active Pending
  • 2023-05-15 TW TW112117922A patent/TW202408551A/zh unknown
  • 2023-05-15 WO PCT/JP2023/018026 patent/WO2023223984A1/ja not_active Ceased
  • 2023-05-15 US US18/865,121 patent/US20250313810A1/en active Pending
  • 2023-05-15 EP EP23807586.5A patent/EP4527394A1/en active Pending

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