METHODS FOR PRODUCING PLATELET-RICH PLASMA

Abstract

The purpose of the invention is to provide a method for producing platelet-rich plasma in which the platelet recovery rate from the blood sample is maintained at a stable high level, platelet-rich plasma produced by such method, and similar. The method for producing platelet-rich plasma includes: step A is centrifuging a blood sample from a mammal at conditions between 100 and 1000 G for 1 to 30 minutes, and then collecting the supernatant; and step B is centrifuging the remaining blood sample after collecting the supernatant in step A at conditions between 100 and 1000 G for 1 to 30 minutes, and then collecting the supernatant to obtain platelet-rich plasma.

Description

Method for producing platelet-rich plasma

[0001] The present invention relates to a method for producing platelet-rich plasma, etc. More specifically, the present invention relates to a method for producing platelet-rich plasma, which has a stable and high recovery rate of platelets from a blood sample.

[0002] Platelet-rich plasma (hereinafter also referred to as "PRP") is a type of platelet-enriched plasma prepared by centrifuging blood. This platelet-rich plasma is rich in various growth factors, which play an effective role in wound healing and tissue regeneration, making it a promising material in the field of regenerative medicine (see, for example, Patent Document 1).

[0003] Furthermore, a method for preparing a blood-derived growth factor-containing composition from mammalian blood and separating platelet-rich plasma from the blood is known, which comprises subjecting the blood to a first centrifugation treatment at 100 to 1,000 G for 1 to 30 minutes to recover an upper layer containing plasma and buffy coat; subjecting the upper layer to a second centrifugation treatment at 1,000 to 2,500 G for 1 to 30 minutes to pellet the platelets and buffy coat components; and removing the supernatant and suspending the platelets and buffy coat components to obtain platelet-rich plasma (Patent Document 2).

[0004] JP 2009-195739 A Japanese Patent No. 7175055 A

[0005] The platelet recovery rate varies considerably depending on the individual differences of the mammal from which the blood sample is collected, and conventional methods for producing platelet-rich plasma have sometimes resulted in a low platelet recovery rate from blood samples in some mammalian individuals. An object of the present invention is to provide a method for producing platelet-rich plasma that stably achieves a high platelet recovery rate from blood samples, and platelet-rich plasma, etc. produced by said method.

[0006] The present inventors have diligently investigated various methods to solve the above-mentioned problems, and have found that the above-mentioned problems can be solved by centrifuging a mammalian blood sample at 100 to 1,000 G for 1 to 30 minutes, recovering the supernatant, and centrifuging the remaining blood sample after the recovery of the supernatant at 100 to 1,000 G for 1 to 30 minutes, and recovering the supernatant, thereby completing the present invention. Furthermore, while clinical PRP preparation kits currently sold by various companies are unable to sufficiently prevent red blood cells and white blood cells from contaminating PRP, the present inventors have found that the PRP production method of the present invention can effectively prevent red blood cells and white blood cells from contaminating PRP, thereby completing the present invention.

[0007] That is, the present invention provides the following inventions, etc.: (1) A method for producing platelet-rich plasma, comprising: step A of centrifuging a mammalian blood sample at 100 to 1,000 G for 1 to 30 minutes and then recovering the supernatant; and step B of obtaining platelet-rich plasma by centrifuging the remaining blood sample after recovery of the supernatant in step A at 100 to 1,000 G for 1 to 30 minutes and then recovering the supernatant; (2) The method according to (1) above, wherein the centrifugation conditions in step A are 100 to 300 G for 5 to 15 minutes and the centrifugation conditions in step B are 100 to 300 G for 5 to 15 minutes; (3) The method according to (1) or (2) above, wherein the mammalian blood sample centrifuged in step A is a blood sample that has been stored at 0 to 35°C for 1 to 120 hours after collection from the mammal; (4) The method according to any one of (1) to (3) above, wherein the mammalian blood sample centrifuged in step A is a blood sample to which an anticoagulant has been added after collection from the mammal; (5) Platelet-rich plasma produced by the method according to any one of (1) to (4) above;

[0008] According to the present invention, it is possible to provide a method for producing platelet-rich plasma which stably achieves a high recovery rate of platelets from a blood sample, and platelet-rich plasma produced by the method.

[0009] This figure shows the platelet recovery rate (%) in the supernatant after centrifugation in the production of platelet-rich plasma in the Examples. The platelet recovery rate (%) is expressed as a percentage (%) of the platelet count in the whole blood of the subject. Furthermore, "EXP1" to "EXP5" on the horizontal axis represent five healthy subjects. In the results for each subject, the leftmost result represents the platelet recovery rate (%) in the supernatant after the first centrifugation, and the second result from the left represents the platelet recovery rate (%) in the supernatant after the second centrifugation. For EXP4 and EXP5, the third result from the left represents the platelet recovery rate (%) in the supernatant after the third centrifugation. In the results for "EXP1" to "EXP3," the rightmost result represents the sum of the recovery rates from the first and second centrifugations (total recovery rate (%)), and in the results for "EX4" and "EX5," the rightmost result represents the sum of the recovery rates from the first to third centrifugations (total recovery rate (%)).

[0010] The present invention includes: [1] a method for producing platelet-rich plasma (hereinafter also referred to as "the production method of the present invention"), comprising: [1] step A of centrifuging a mammalian blood sample at 100 to 1,000 G for 1 to 30 minutes, and then recovering the supernatant; and step B of obtaining platelet-rich plasma by centrifuging the remaining blood sample after recovery of the supernatant in step A at 100 to 1,000 G for 1 to 30 minutes, and then recovering the supernatant; [2] platelet-rich plasma produced by the production method of the present invention (hereinafter also referred to as "the platelet-rich plasma of the present invention"); and the like.

[0011] <Production Method of the Present Invention> The production method of the present invention is not particularly limited as long as it is a method for producing platelet-rich plasma, comprising: step A of centrifuging a mammalian blood sample at 100 to 1,000 G for 1 to 30 minutes and then recovering the supernatant; and step B of centrifuging the remaining blood sample after recovery of the supernatant in step A at 100 to 1,000 G for 1 to 30 minutes and then recovering the supernatant, thereby obtaining platelet-rich plasma.

[0012] (Step A) Step A is not particularly limited as long as it is a step of centrifuging a mammalian blood sample at 100 to 1000 G for 1 to 30 minutes and then recovering the supernatant.

[0013] The "mammal" includes, for example, humans and non-human mammals, with humans being preferred. The non-human mammals include, for example, mice, rats, guinea pigs, dogs, cats, monkeys, rabbits, sheep, horses, pigs, etc.

[0014] The "blood sample" is not particularly limited as long as it is a blood sample containing platelets, and whole blood is preferred. A blood sample can be collected from a mammal by a conventional method.

[0015] The "blood sample" to be centrifuged in step A is preferably a blood sample to which an anticoagulant has been added after collection from a mammal. Although an anticoagulant may be added to the collected blood sample after collection, it is preferable to collect the blood sample in a container such as a blood collection tube or blood collection bag that already contains an anticoagulant. Examples of the "anticoagulant" include chelating agents that inhibit blood coagulation by coordinating calcium ions, which are a factor in blood coagulation, and antithrombin agents containing heparin that inhibit thrombin activity, which is a factor in blood coagulation. Among these, chelating agents are preferred. Examples of anticoagulants that are chelating agents include one or more selected from the group consisting of citric acid, EDTA (ethylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), DCTA (1,2-diaminocyclohexanetetraacetic acid), EGTA (ethylene glycol bis-2-aminoethyl ether tetraacetic acid), oxalic acid, and salts thereof. Among these, citric acid or a salt thereof is preferred. Examples of the salt include alkali metal salts, and more preferably sodium salts. The anticoagulant may be added as a solid, or may be added as a solution (anticoagulant solution) prepared by dissolving the anticoagulant in a solvent such as pure water (preferably a physiologically acceptable solvent). Specific examples of aqueous solutions containing an anticoagulant include ACD-A solution (aqueous solution containing sodium citrate hydrate, citric acid hydrate, and glucose), CPD solution (aqueous solution containing sodium citrate hydrate, citric acid hydrate, glucose, and sodium dihydrogen phosphate hydrate), and CPDA-1 solution (aqueous solution containing sodium citrate hydrate, citric acid hydrate, glucose, sodium dihydrogen phosphate hydrate, and adenine), with ACD-A solution being preferred. A single anticoagulant may be used, or two or more may be used in combination. Commercially available anticoagulants and anticoagulant solutions may be used.

[0016] The concentration of the anticoagulant in a blood sample to which an anticoagulant (or anticoagulant solution) has been added is not particularly limited as long as the effects of the present invention are obtained. For example, when the anticoagulant is citric acid or a salt thereof, the concentration in the blood sample to which the anticoagulant (or anticoagulant solution) has been added is, for example, 0.2 to 3 w / v %, preferably 0.5 to 2 w / v %, in terms of citric acid. Similarly, when the anticoagulant is EDTA or a salt thereof, the EDTA-equivalent concentration is, for example, 0.2 to 3 w / v%, preferably 0.5 to 2 w / v%; when the anticoagulant is DTPA or a salt thereof, the DTPA-equivalent concentration is, for example, 0.2 to 3 w / v%, preferably 0.5 to 2 w / v%; when the anticoagulant is DCTA or a salt thereof, the DCTA-equivalent concentration is, for example, 0.2 to 3 w / v%, preferably 0.5 to 2 w / v%; when the anticoagulant is EGTA or a salt thereof, the EGTA-equivalent concentration is, for example, 0.2 to 3 w / v%, preferably 0.5 to 2 w / v%; and when the anticoagulant is oxalic acid or a salt thereof, the oxalic acid-equivalent concentration is, for example, 0.2 to 3 w / v%, preferably 0.5 to 2 w / v%. When two or more anticoagulants are used in combination, the total concentration can be, for example, 0.2 to 3 w / v %, preferably 0.5 to 2 w / v %.

[0017] The "blood sample" to be centrifuged in step A does not need to be stored for, for example, 1 hour or less after collection from the mammal. However, since a longer storage time tends to decrease the platelet recovery rate, from the viewpoint of enjoying the full benefits of the present invention, it is preferable that the "blood sample" to be centrifuged in step A be a blood sample that has been stored for, for example, 1 hour or more after collection from the mammal. The lower limit of the storage time for a blood sample stored for 1 hour or more is preferably 3 hours or more, 8 hours or more, 12 hours or more, 18 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, 60 hours or more, 72 hours or more, 84 hours or more, 96 hours or more, 108 hours or more, or 120 hours or more, and the upper limit is preferably 14 days or less, 12 days or less, 10 days or less, 8 days or less, or 6 days or less. These lower and upper limits can be combined in any desired manner. The temperature at which the blood sample is stored is not particularly limited, but examples include 0 to 35°C, 0 to 30°C, 0 to 25°C, 0 to 20°C, 0 to 15°C, 0 to 10°C, and 0 to 8°C.

[0018] The conditions for centrifugation in step A are not particularly limited as long as the centrifugation is performed at 100 to 1000 G for 1 to 30 minutes. The centrifugal force under the above conditions is not particularly limited as long as it is 100 to 1000 G, but is preferably 100 to 900 G, 100 to 800 G, 100 to 700 G, 100 to 600 G, 100 to 500 G, 100 to 400 G, 100 to 300 G, 100 to 250 G, 150 to 900 G, 150 to 800 G, 150 to 700 G, 150 to 600 G, 150 to 500 G, 150 to 600 G, 150 to 500 G, 150 to 800 G, 150 to 900 G, 150 to 800 G, 150 to 700 G, 150 to 600 G, 150 to 500 G, 150 to 10 ... Examples include 0 to 400G, 150 to 300G, and 150 to 250G, more preferably 100 to 500G, 100 to 400G, 100 to 300G, 100 to 250G, 150 to 500G, 150 to 400G, 150 to 300G, and 150 to 250G, and even more preferably 100 to 300G, 100 to 250G, 150 to 300G, and 150 to 250G. The centrifugation time under the above conditions is not particularly limited as long as it is 1 to 30 minutes, but is preferably 2 to 25 minutes, 3 to 25 minutes, 4 to 25 minutes, 5 to 25 minutes, 2 to 20 minutes, 3 to 20 minutes, 4 to 20 minutes, or 5 to 20 minutes, and more preferably 5 to 18 minutes, 6 to 18 minutes, 7 to 18 minutes, 5 to 15 minutes, 6 to 15 minutes, 7 to 15 minutes, 5 to 13 minutes, 6 to 13 minutes, or 7 to 13 minutes.

[0019] The blood sample can be centrifuged using a commercially available centrifugal separator.

[0020] In step A, the method for recovering the supernatant after centrifugation is not particularly limited, and examples include methods for recovering all of the plasma fractions (platelet-rich plasma fraction and platelet-poor plasma fraction) or only the platelet-rich plasma fraction.

[0021] When recovering the supernatant, any volume can be recovered so as to contain as few fractions containing red blood cells as possible (for example, visually). Specifically, for example, the volume ratio of the recovered supernatant to the total volume of the liquid after centrifugation in step A is preferably 1:0.1 to 0.5, more preferably 1:0.2 to 0.5, and even more preferably 1:0.3 to 0.5.

[0022] (Step B) Step B is not particularly limited as long as it is a step of obtaining platelet-rich plasma by centrifuging the residual blood sample after supernatant recovery in step A at 100 to 1000 G for 1 to 30 minutes and then recovering the supernatant. The "residual blood sample" centrifuged in step B may be the blood sample remaining after the supernatant recovery in step A (i.e., the residual blood sample) itself, or may be a residual blood sample obtained by subjecting the residual blood sample to some additional processing, as long as the effects of the present invention are obtained. The platelet recovery rate varies considerably depending on factors such as individual differences between mammals from which the blood sample is collected. By including step B in addition to step A in the production method of the present invention, the influence of individual differences between mammals from which the blood sample is collected can be reduced, and a stably high platelet recovery rate can be maintained.

[0023] The conditions for centrifugation in step B are not particularly limited as long as the centrifugation is performed at 100 to 1000 G for 1 to 30 minutes. The centrifugal force under the above conditions is not particularly limited as long as it is 100 to 1000 G, but is preferably 100 to 900 G, 100 to 800 G, 100 to 700 G, 100 to 600 G, 100 to 500 G, 100 to 400 G, 100 to 300 G, 100 to 250 G, 150 to 900 G, 150 to 800 G, 150 to 700 G, 150 to 600 G, 150 to 500 G, 150 to 600 G, 150 to 500 G, 150 to 800 G, 150 to 900 G, 150 to 800 G, 150 to 700 G, 150 to 600 G, 150 to 500 G, 150 to 10 ... Examples include 0 to 400G, 150 to 300G, and 150 to 250G, more preferably 100 to 500G, 100 to 400G, 100 to 300G, 100 to 250G, 150 to 500G, 150 to 400G, 150 to 300G, and 150 to 250G, and even more preferably 100 to 300G, 100 to 250G, 150 to 300G, and 150 to 250G. The centrifugation time under the above conditions is not particularly limited as long as it is 1 to 30 minutes, but is preferably 2 to 25 minutes, 3 to 25 minutes, 4 to 25 minutes, 5 to 25 minutes, 2 to 20 minutes, 3 to 20 minutes, 4 to 20 minutes, or 5 to 20 minutes, and more preferably 5 to 18 minutes, 6 to 18 minutes, 7 to 18 minutes, 5 to 15 minutes, 6 to 15 minutes, 7 to 15 minutes, 5 to 13 minutes, 6 to 13 minutes, or 7 to 13 minutes. The centrifugation conditions in step A and the centrifugation conditions in step B may be the same or different.

[0024] In step B, the method for recovering the supernatant after centrifugation is not particularly limited, but a preferred example is a method for recovering the supernatant containing a small amount of white blood cell components and / or red blood cell components. Such a supernatant contains platelets at a relatively high concentration.

[0025] When recovering the supernatant, any volume can be recovered so as to contain as few fractions containing red blood cells as possible (for example, visually). Specifically, for example, the volume ratio of the recovered supernatant to the total volume of the liquid after centrifugation in step B is preferably 1:0.1 to 0.9, more preferably 1:0.1 to 0.6, and even more preferably 1:0.1 to 0.4.

[0026] (Optional Steps) The production method of the present invention may include only steps A and B, or may include other optional steps. Such optional steps include step C of obtaining platelet-rich plasma by centrifuging the residual blood sample after supernatant recovery in step B at 100 to 1,000 G for 1 to 30 minutes and then recovering the supernatant. The platelet recovery rate varies considerably depending on individual differences between mammals from which the blood sample is collected. Step C can be preferably performed from the viewpoint of further improving the platelet recovery rate when steps A and B are not particularly high. The centrifugation conditions in steps A and B may be the same as or different from the centrifugation conditions in step C. Furthermore, step C may be further repeated using the "residual blood sample after supernatant recovery" in step C instead of the "residual blood sample after supernatant recovery in step B." The total number of centrifugation cycles when steps A to C or step C are further repeated is not particularly limited, and may be, for example, 3 to 6 times or 3 to 4 times. The centrifugation conditions when step C is repeated may be the same or different.

[0027] An optional step other than the above step C includes step D, in which blood cells other than platelets are removed from the platelet-rich plasma obtained in step B or step C (or, if step C is repeated, step C performed last). Examples of methods for removing blood cells other than platelets from platelet-rich plasma include filtering. Note that, since the production method of the present invention can efficiently prepare platelet-rich plasma with little contamination by blood cells such as leukocytes, the treatment in the above step D (e.g., filtering) may not be necessary.

[0028] An optional step other than the above-mentioned step C or step D includes step E, which calcium-stimulates platelets in the platelet-rich plasma obtained in step B or step C (or, if step C is repeated, step C performed last) or step D. Calcium-stimulating the platelets of the present invention results in the secretion of more cytokines from the platelets, thereby enabling platelet-rich plasma with improved anti-inflammatory effects and the like to be obtained. The calcium stimulation method is not particularly limited, but includes a method in which platelets in the platelet-rich plasma of the present invention are incubated in a solution having a calcium concentration of, for example, 0.01 to 100 mM, preferably 0.5 to 50 mM, and more preferably 5 to 20 mM. The incubation time is not particularly limited, but includes, for example, 0.1 minute to 3 hours, preferably 10 to 20 minutes. The calcium-containing compound used for calcium stimulation is not particularly limited, but includes calcium salts such as calcium chloride. Furthermore, the timing of calcium stimulation is not particularly limited, but includes, for example, administration of the platelet-rich plasma of the present invention to a mammal (preferably a human) or the like having a disease, such as within 5 hours, 3 hours, 1 hour, or 30 minutes prior to administration.

[0029] An optional step other than the above step C, step D, or step E includes step F, which is freezing the platelet-rich plasma obtained in step B or step C (or, if step C is repeated, step C performed last), step D, or step E. The freezing may be a freezing treatment other than freeze-drying, or may be a freeze-drying treatment. Frozen platelet-rich plasma is preferred because it can be stored for a longer period of time.

[0030] When the production method of the present invention has optional steps, the production method of the present invention may have, in addition to the essential steps of step A and step B, one or more (two, three, or four) optional steps selected from the group consisting of step C, step D, step E, and step F.

[0031] (Total Platelet Recovery Rate) The total platelet recovery rate (%) in the production method of the present invention is not particularly limited, and the lower limit can be, for example, 40% or more, preferably 45% or more, 50% or more, 55% or more, or 60% or more, and the upper limit can be, for example, 100% or less, 95% or less, 90% or less, 85% or less, or 80% or less. These lower and upper limits can be combined arbitrarily. As used herein, the term "total platelet recovery rate (%)" refers to the ratio (%) of the total number of platelets recovered by the production method of the present invention to the total number of platelets in the blood sample in step A.

[0032] <Platelet-Rich Plasma of the Present Invention> The platelet-rich plasma of the present invention is not particularly limited as long as it is platelet-rich plasma produced by the production method of the present invention.

[0033] It has been pointed out that the presence of red blood cells in platelet-rich plasma (PRP) preparations can lead to adverse effects such as microcirculatory dysfunction, vascular damage, tissue injury, and degenerative damage to joints (Biomedicines 2023, 11, 2425. https: / / doi.org / 10.3390 / biomedicines11092425). The red blood cell count in the platelet-rich plasma of the present invention is not particularly limited, but examples include 10,000 / μL or less, 1,000 / μL or less, and preferably 500 / μL or less, 300 / μL or less, 200 / μL or less, 100 / μL or less, and 50 / μL or less. Furthermore, the white blood cell count in the platelet-rich plasma of the present invention is not particularly limited, but examples include 500 / μL or less, and preferably 300 / μL or less, 200 / μL or less, and 100 / μL or less.

[0034] The concentration of vascular endothelial growth factor (VEGF) in the platelet-rich plasma of the present invention is not particularly limited, but is preferably 100 pg / mL, more preferably 300 pg / mL or more, even more preferably 700 pg / mL or more, and even more preferably 1000 pg / mL or more. The upper limit varies depending on the individual differences of the mammal from which the blood sample was collected, but is, for example, 30000 pg / mL.

[0035] The platelet-rich plasma of the present invention may be contained in a container. The material and shape of the container are not particularly limited, and examples thereof include cryotubes, cryovials, freezing bags, and infusion bags.

[0036] The platelet-rich plasma of the present invention can be used for any purpose, such as blood transfusion and regenerative medicine.

[0037] The present invention will be specifically described based on the following examples, but the present invention is not limited to these examples.

[0038] Test 1. [Confirmation of platelet recovery rate by the production method of the present invention] The following test was carried out to confirm the platelet recovery rate by the production method of the present invention.

[0039] Five 20 mL syringes were filled with 3 mL of acid citrate dextrose solution (ACD solution) as an anticoagulant. Approximately 20 mL of blood was collected from each of five healthy volunteers ("EXP1" to "EXP5") using the syringes described above, and the whole blood was dispensed into polypropylene tubes. The platelet count in each tube was then measured.

[0040] The five polypropylene tubes containing whole blood were placed in a centrifuge and centrifuged at 200 G for 10 minutes to recover the supernatant plasma (approximately 5 mL: a recovered volume ratio of 1:0.4 based on the total volume of the liquid after centrifugation). The number of platelets in the recovered supernatant was counted using a hematology analyzer, and the recovery rate (%) relative to the number of platelets in the whole blood was calculated. The results are shown in Figure 1 as the "first run" results.

[0041] As can be seen from FIG. 1, the platelet recovery rate after the first centrifugation was 20 to 50%.

[0042] The remaining blood (residual blood) sample from which the supernatant was recovered after the first centrifugation was centrifuged for 10 minutes at 200 G as a second centrifugation, and the supernatant plasma (approximately 3 mL: a volume ratio of 1:0.15 based on the total volume of the liquid after the second centrifugation) was recovered. The number of platelets in the recovered supernatant was counted using a hematology analyzer, and the recovery rate (%) relative to the number of platelets in the whole blood was calculated. The results are shown in Figure 1 as the "second" results.

[0043] As can be seen from Figure 1, the platelet recovery rate after the second centrifugation was 15 to 40%. Furthermore, when the recovery rates after the first and second centrifugations were combined, a high recovery rate of approximately 50 to 80% was obtained.

[0044] For EXP4 and EX5, the remaining blood (residual blood) samples from which the supernatant was recovered after the second centrifugation were centrifuged for a third time at 200 G for 10 minutes to recover the supernatant plasma (approximately 2 mL: a recovered volume ratio of 1:0.13 based on the total volume of the liquid after the third centrifugation). The platelet count in the recovered supernatant was measured using a hematology analyzer, and the recovery rate (%) relative to the platelet count in the whole blood was calculated. The results are shown in Figure 1 as the "third" results.

[0045] As can be seen from Figure 1, the platelet recovery rate after the third centrifugation was 4 to 20%, which indicates that when the total recovery rate after the first and second centrifugations is low, a slightly larger amount of platelets can be recovered by the third centrifugation.

[0046] Furthermore, for EXP1 to 3, the total recovery rate is the sum of the recovery rates from the first and second centrifugation, and for EXP4 to 5, the total recovery rate is the sum of the recovery rates from the first, second, and third centrifugation, and these are shown in Figure 1. These results demonstrate that the method for producing platelet-rich plasma of the present invention enables platelets to be recovered from blood samples at a high recovery rate.

[0047] Test 2. [Confirmation of red blood cell and white blood cell counts in platelets produced by the production method of the present invention] It has been pointed out that the presence of red blood cells in platelet-rich plasma (PRP) preparations can lead to adverse effects such as microcirculatory dysfunction, vascular damage, tissue damage, and degenerative damage to joints (Biomedicines 2023, 11, 2425. https: / / doi.org / 10.3390 / biomedicines11092425). Therefore, the following test was conducted to confirm the red blood cell and white blood cell counts in the platelet-rich plasma produced by the production method of the present invention.

[0048] Blood samples were collected from 15 healthy volunteers and centrifuged twice according to the method described in Test 1 above. The number of red blood cells and white blood cells contained in each PRP with a platelet recovery rate of 75% or higher was measured using an automatic blood cell counter CBC LC-661 (manufactured by Fukuda Denshi Co., Ltd.). The results are shown in Table 1.

[0049]

[0050] Table 1 shows that the PRP produced by the production method of the present invention has extremely low numbers of contaminating red blood cells and white blood cells. This shows that the production method of the present invention is significantly superior as a PRP production method even in an embodiment in which centrifugation is performed twice (i.e., an embodiment having steps A and B but not step C).

[0051] According to the present invention, it is possible to provide a method for producing platelet-rich plasma which stably achieves a high recovery rate of platelets from a blood sample, and platelet-rich plasma produced by the method.

Claims

1. A method for producing platelet-rich plasma, comprising: step A of centrifuging a mammalian blood sample at 100 to 1,000 G for 1 to 30 minutes and then recovering the supernatant; and step B of centrifuging the remaining blood sample after recovery of the supernatant in step A at 100 to 1,000 G for 1 to 30 minutes and then recovering the supernatant to obtain platelet-rich plasma.

2. The method according to claim 1, wherein the centrifugation conditions in step A are 100 to 300 G for 5 to 15 minutes, and the centrifugation conditions in step B are 100 to 300 G for 5 to 15 minutes.

3. The method according to claim 1, wherein the mammalian blood sample centrifuged in step A is a blood sample that has been stored at 0 to 35° C. for 1 to 120 hours after being collected from the mammal.

4. The method according to claim 1, wherein the mammalian blood sample centrifuged in step A is a blood sample to which an anticoagulant has been added after being drawn from the mammal.

5. Platelet-rich plasma produced by the method according to any one of claims 1 to 4.

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