WO2022181798A1 - 成長因子混合物を調整する方法 - Google Patents

成長因子混合物を調整する方法 Download PDF

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WO2022181798A1
WO2022181798A1 PCT/JP2022/008038 JP2022008038W WO2022181798A1 WO 2022181798 A1 WO2022181798 A1 WO 2022181798A1 JP 2022008038 W JP2022008038 W JP 2022008038W WO 2022181798 A1 WO2022181798 A1 WO 2022181798A1
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platelet
rich plasma
growth factor
centrifugation
blood
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PCT/JP2022/008038
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English (en)
French (fr)
Japanese (ja)
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景真 上野
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株式会社同仁がん免疫研究所
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Priority to JP2022541701A priority Critical patent/JP7313098B2/ja
Publication of WO2022181798A1 publication Critical patent/WO2022181798A1/ja

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    • 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
    • A61K35/16—Blood plasma; Blood serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present disclosure relates to methods for preparing growth factor mixtures.
  • PRP therapy in which plasma containing a large number of platelets (platelet-rich plasma, PRP) is separated from blood and injected into the affected area.
  • PRP therapy uses platelet-derived growth factor (PDGF), transforming growth factor- ⁇ (TGF- ⁇ ), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF) contained in platelets. ) and other growth factors to enhance healing power.
  • PDGF platelet-derived growth factor
  • TGF- ⁇ transforming growth factor- ⁇
  • FGF fibroblast growth factor
  • VEGF vascular endothelial growth factor
  • EGF epidermal growth factor
  • the growth factor mixture obtained by further concentrating and activating this platelet-rich plasma and recovering platelet-derived components from the concentrated and activated platelet-rich plasma is highly safe because it is decellularized. It is highly convenient because it is already concentrated and activated. Therefore, it is expected as a promising material in the field of regenerative medicine.
  • the present disclosure provides: (Item 1) A method for preparing a growth factor mixture from blood, comprising: A step of subjecting the blood to a first centrifugation treatment and recovering the supernatant; subjecting the supernatant to a second centrifugation process to obtain platelet-rich plasma; a step of subjecting the platelet-rich plasma to a treatment for activating platelets; obtaining a growth factor mixture from the platelet-rich plasma with activated platelets; A method, including (Item 1a) The method according to any of the preceding items, wherein the supernatant contains leukocytes. (Item 1b) The method according to any one of the above items, wherein the platelet-rich plasma is leukocyte-containing platelet-rich plasma.
  • (Item 2) The method according to any one of the above items, wherein the first centrifugation treatment and the second centrifugation treatment are performed under the same conditions.
  • the first centrifugation treatment and the second centrifugation treatment are performed at about 100 G to about 3000 G for about 2 to about 30 minutes at room temperature.
  • Method. The method according to any one of the above items, wherein the first centrifugation treatment and the second centrifugation treatment are performed at about 940 G for about 15 minutes at about 24°C.
  • the activating treatment includes adding an agent that activates platelets to the platelet-rich plasma.
  • the platelet-activating agent comprises calcium chloride.
  • the growth factor mixture is not lyophilized.
  • the step of obtaining the growth factor mixture comprises removing cells from the platelet-rich plasma in which platelets have been activated.
  • removing cells from the platelet-rich plasma includes filtering.
  • a method comprising administering an effective amount.
  • (Item A2) A method for treating a disease, disorder or condition in a subject, the method comprising: obtaining blood from the subject; a step of subjecting the blood to a first centrifugation treatment and recovering the supernatant; subjecting the supernatant to a second centrifugation process to obtain platelet-rich plasma; a step of subjecting the platelet-rich plasma to a treatment for activating platelets; obtaining a growth factor mixture from the platelet-rich plasma with activated platelets; administering to said subject a therapeutically effective amount of said growth factor mixture.
  • a method for treating a disease, disorder or condition in a subject comprising administering to the subject a therapeutically effective amount of a growth factor mixture, the growth factor mixture comprising: A step of subjecting the blood to a first centrifugation treatment and recovering the supernatant; subjecting the supernatant to a second centrifugation process to obtain platelet-rich plasma; a step of subjecting the platelet-rich plasma to a treatment for activating platelets; obtaining a growth factor mixture from the platelet-rich plasma with activated platelets; A method prepared by a method comprising: (Item A4) A method for treating a disease, disorder or condition in a subject, the method comprising: obtaining blood from the subject; a step of subjecting the blood to a first centrifugation treatment and recovering the supernatant; subjecting the supernatant to a second centrifugation process to obtain platelet-rich plasma; a step of subjecting the platelet-rich plasma to a treatment for activating platelets; and administering to
  • (Item A5) A method for treating a disease, disorder, or condition in a subject comprising administering to the subject a therapeutically effective amount of platelet-rich plasma, the platelet-rich plasma comprising: A step of subjecting the blood to a first centrifugation treatment and recovering the supernatant; subjecting the supernatant to a second centrifugation process to obtain platelet-rich plasma; a step of subjecting the platelet-rich plasma to a treatment for activating platelets; A method prepared by a method comprising: (Item A6) A method according to any one of items A1-A5 comprising the features of the method according to any one of items 1-12a. (Item B1) A growth factor mixture prepared by the method according to any one of the preceding items.
  • a blood-derived growth factor mixture comprising: A step of subjecting the blood to a first centrifugation treatment and recovering the supernatant; subjecting the supernatant to a second centrifugation process to obtain platelet-rich plasma; a step of subjecting the platelet-rich plasma to a treatment for activating platelets; obtaining a growth factor mixture from the platelet-rich plasma with activated platelets;
  • a growth factor mixture prepared by a method comprising: (Item B3) A growth factor mixture prepared by the method of any of the preceding items for treating a disease, disorder or condition of interest.
  • a blood-derived growth factor mixture for treating a disease, disorder or condition in a subject comprising: A step of subjecting the blood to a first centrifugation treatment and recovering the supernatant; subjecting the supernatant to a second centrifugation process to obtain platelet-rich plasma; a step of subjecting the platelet-rich plasma to a treatment for activating platelets; obtaining a growth factor mixture from the platelet-rich plasma with activated platelets;
  • a group of growth factors can be efficiently recovered from platelet-rich plasma and activated, which is useful in the medical field.
  • it since it is acellular, it is possible to avoid legal restrictions such as the Regenerative Medicine Safety Assurance Act, which is highly convenient.
  • FIG. 1 is a schematic diagram showing a series of flows from blood collection to production of a growth factor mixture in one embodiment of the present disclosure.
  • FIG. 2 is a schematic diagram showing a series of flows from blood collection to production of a growth factor mixture in another embodiment of the present disclosure.
  • growth factor mixture refers to any mixture of growth factors, typically including platelet-derived cytokines and other growth factors.
  • a mixture of growth factors is not particularly limited, as long as two or more elements coexist in a natural state, and does not have to be produced by an operation of actively mixing. good too.
  • platelet-rich plasma refers to a plasma concentrate containing more platelets than normal plasma, and can be obtained by centrifuging plasma in blood.
  • the concentration of platelets is about one or more times that of blood (whole blood), typically about two to about seven times.
  • Platelet-rich plasma has a concentration of, for example, about 10 ⁇ 10 4 / ⁇ L or more, about 25 ⁇ 10 4 / ⁇ L or more, about 50 ⁇ 10 4 / ⁇ L or more, or preferably about 100 ⁇ 10 4 / ⁇ L or more. of platelets.
  • platelet-rich plasma is also typically rich in growth factors such as PDGF, TGF- ⁇ , VEGF, EGF, FGF, IGF, or HGF.
  • the term "platelet-rich plasma containing leukocytes" refers to the above-described platelet-rich plasma containing leukocytes.
  • activation of platelets refers to a series of reactions that occur in platelets during bleeding, equivalent reactions, or partial reactions. A large number of long projections appear and form a shape like a confetti, or growth factors produced in platelets are released outside platelets, or growth factors stored inside platelets are released outside platelets. Say things.
  • a method of preparing a growth factor mixture from blood comprising the steps of subjecting the blood to a first centrifugation treatment and recovering the supernatant; a step of centrifuging to obtain platelet-rich plasma; a step of subjecting the platelet-rich plasma to platelet activation processing; and a step of obtaining a growth factor mixture from the platelet-rich plasma with activated platelets.
  • a method comprising: The method of the present disclosure is useful as a technique for providing promising materials in the field of regenerative medicine.
  • the supernatant can contain leukocytes.
  • the leukocyte-containing supernatant preferably contains leukocytes at a concentration of about 1.5 times or more than whole blood, but is not so limited.
  • the platelet-rich plasma can be leukocyte-containing platelet-rich plasma.
  • the leukocyte-containing platelet-rich plasma preferably contains leukocytes at a concentration of about 5 times or more that of whole blood, but is not limited thereto.
  • the first centrifugation treatment for blood and the second centrifugation treatment for the supernatant obtained by this first centrifugation treatment are preferably performed under the same conditions.
  • the first or second centrifugation process can be about 100 to about 3000G centrifugation, preferably about 500 to about 1000G, or about 940G.
  • Treatment times can be from about 2 to about 30 minutes, preferably from about 5 to about 20 minutes, or about 15 minutes.
  • Centrifugation of blood and supernatant is preferably performed at room temperature, for example, at about 20°C to about 27°C, more preferably at about 24°C.
  • the supernatant By subjecting the blood (whole blood) to the first centrifugation treatment as described above, the supernatant can be separated, and by subjecting the supernatant to the second centrifugation treatment , platelet-rich plasma can be separated.
  • the first centrifugation process and the second centrifugation process can also be performed multiple times as needed.
  • the step of subjecting the platelet-rich plasma collected as described above to a treatment for activating platelets is performed by adding an agent that activates platelets to the platelet-rich plasma.
  • an agent that activates platelets can also When platelets are activated in this manner, a large number of platelet-derived growth factors are released and activated.
  • the treatment to activate platelets may be drug-free.
  • a method using cold stimulation a method using ultrasonic treatment, a method of attaching platelets to a substrate, and the like may be used.
  • the cold stimulation temperature in this case may be about 0° C. or higher, preferably about 4° C. or higher, and the like.
  • Agents that activate platelets are not particularly limited, but calcium ion source compounds such as calcium chloride, calcium phosphate, calcium lactate, and calcium carbonate can be used, and platelets are activated by the presence of calcium ions. be.
  • the agent that activates platelets can be added as a solution, and its concentration is not particularly limited, but for example about 1% by mass or more, about 1.5% by mass or more, about It can be 2 wt% or more, about 2.5 wt% or more. In one embodiment, the amount added is not particularly limited. about 0.2 times or more, about 0.25 times or more, about 0.3 times or more by volume.
  • the step of obtaining a growth factor mixture from the platelet-rich plasma with activated platelets can include a process of removing cells from the platelet-activated platelet-rich plasma.
  • removal of cells is not particularly limited as long as it can remove cells from platelet-rich plasma, and filtering treatment can be preferably used.
  • the filtering process removes various cells such as leukocytes, platelets, and erythrocytes contained in the platelet-rich plasma, and the membrane pore size can be appropriately set according to the size of the cells to be removed.
  • filters with multiple membrane pore sizes can be used as needed, and these can be combined and filtered multiple times to remove cells other than platelets.
  • a membrane filter with a pore size of about 5 ⁇ m or less, about 1 ⁇ m or less, about 0.7 ⁇ m or less, about 0.5 ⁇ m or less, about 0.3 ⁇ m or less can be used, and filters cells to be removed. Anything that can be done is good.
  • filtering can be performed by centrifugation, and various cells such as platelets and leukocytes can be removed by centrifuging the platelet-rich plasma using the filter described above.
  • the conditions for the centrifugation treatment the same conditions as those for the first centrifugation treatment and the second centrifugation treatment can be employed.
  • the growth factor mixture and the cell-free plasma in the preceding stage thereof contain substantially no cells (particularly platelets) or no cells at all can be demonstrated, for example, by confirming no cells larger than 1 ⁇ m using a cell counter. . Alternatively, it may be verified by not observing platelets using a hemocytometer, a hemocytometer, or the like. It is sufficient if it can be confirmed by any means that it does not contain platelets.
  • the growth factor mixture can be made substantially cell-free. Therefore, conventional platelet-rich plasma contains cells and is subject to various legal restrictions. However, the growth factor mixture of the present disclosure does not contain cells, so it can be used as a material for regenerative medicine without restrictions. can be used.
  • the supernatant containing fibrinogen and/or albumin can also be removed after performing the second centrifugation treatment as described above.
  • precipitated (separated) blood cells can be suspended in a small amount of PBS (phosphate-buffered saline) to obtain a blood cell suspension (platelet-rich plasma).
  • a platelet-activating agent, such as those described above, is then added to this suspension.
  • fibrinogen is less than about 10 mg/mL, less than about 7 mg/mL in the growth factor mixture finally obtained by the method of the present disclosure by removing fibrinogen and/or albumin as described above. , less than about 5 mg/mL, less than about 4 mg/mL, less than about 3 mg/mL, less than about 2 mg/mL, less than about 1 mg/mL, and albumin is substantially removed at levels less than about 50 mg/mL, about It can be substantially removed at levels less than 40 mg/mL, less than about 37 mg/mL, less than about 30 mg/mL, less than about 20 mg/mL, or less than about 10 mg/mL.
  • the growth factor mixture obtained by removing cells as described above can be used without being freeze-dried.
  • the growth factor mixture obtained by the method of the present disclosure can contain growth factors contained in normal platelet-rich plasma.
  • growth factors include PDGF, TGF- ⁇ , VEGF, EGF, FGF, IGF, HGF and the like.
  • a growth factor mixture of the present disclosure can contain at least these factors.
  • various components can be included, for example, in the following ranges.
  • PDGF about 1 to about 400 ng/mL (PDGF-BB)
  • TGF- ⁇ about 2000 to about 12000 pg/mL
  • EGF about 5 to about 60 pg/mL
  • FGF about 500 to about 3200 pg/mL
  • FGF-4 VEGF: about 10 to about 2000 pg/mL
  • IGF about 250 to about 6000 pg/mL
  • PDGF about 50 to about 400 ng/mL (PDGF-BB)
  • TGF- ⁇ about 2000 to about 12000 pg/mL
  • EGF about 5 to about 60 pg/mL
  • FGF about 500 to about 3200 pg/mL
  • FGF-4 VEGF: about 10 to about 800 pg/mL
  • IGF about 250 to about 6000 pg/mL
  • the growth factor mixture obtained by the method of the present disclosure may be free or substantially free of fibrinogen and/or albumin.
  • Fibrinogen about 0 to about 10 mg/mL, about 0 to about 7 mg/mL, or about 0 to about 5 mg/mL
  • Albumin about 0 to about 50 mg/mL, about 0 to about 40 mg/mL, about 0 to about 30 mg/mL, about 0 to about 20 mg/mL, or about 0 to about 10 mg/mL
  • the above about 0 can be the lowest detectable value at the limit of detection, such as about 0.0001 mg/mL.
  • fibrinogen and/or albumin are included, the following concentrations are acceptable.
  • Fibrinogen about 0.0001 mg/mL to about 10 mg/mL, about 0.0001 mg/mL to about 7 mg/mL, or about 0.0001 mg/mL to about 5 mg/mL
  • Albumin about 0.0001 mg/mL to about 50 mg/mL, about 0.0001 mg/mL to about 40 mg/mL, about 0.0001 mg/mL to about 30 mg/mL, about 0.0001 mg/mL to about 20 mg/mL, or from about 0.0001 mg/mL to about 10 mg/mL
  • the tissue to be administered with the growth factor mixture obtained by the method of the present disclosure is not particularly limited, and can be administered to joints and muscle tendons.
  • the growth factor mixtures of the present disclosure can be stored at - about 80°C to about 25°C, and can be stored at room temperature from about 20°C to about 25°C when used.
  • the sequential flow of the method of the present disclosure may be as follows. That is, after blood collection, the first centrifugation is performed at about 2000 rpm (980 g) for 15 minutes at 20-25° C., the upper 1/2-2/3 of the serum is discarded, and the middle layer is collected. At this time, erythrocytes can be excluded using a lymphocyte separation liquid, a BD Vacutainer CPT blood collection tube, or the like. After that, about 10 ml is collected in a centrifuge tube, diluted by adding PBS so as to reduce fibrin, and subjected to a second centrifugation at about 2000 rpm (980 g) for 15 minutes at 20-25°C. Discard the supernatant, add PBS or 2% CaCl 2 if necessary, mix well, and activate platelets and release growth factors at 20-25°C.
  • the solution can then be further centrifuged at about 2000 rpm (980 g) for 2 minutes at 20-25° C. if not freeze-thawed.
  • filter treatment or the like may be performed to remove cells other than platelets (white blood cells and red blood cells) before freezing. Freeze at -80°C for 30 minutes or more. After thawing, it can be centrifuged at 14000 rpm, 5 minutes, 20-25°C.
  • the supernatant after centrifugation is filtered through a filter of about 0.45 ⁇ m, and VEGF, fibrinogen, albumin, etc. can be measured in this solution. It can also be confirmed to be free of cells and growth factor mixtures of the present disclosure.
  • a growth factor mixture or platelet-rich plasma of the present disclosure can be used to treat a disease, disorder, or condition in a subject.
  • a growth factor mixture or platelet-rich plasma of the present disclosure can be prepared using blood obtained from a subject and administered to the subject in a therapeutically effective amount to treat a disease, disorder or condition in that subject.
  • treatment with the growth factor mixture or platelet-rich plasma of the present disclosure is adjusted according to the state and type of disease, patient's condition, patient's wishes, etc. can be done.
  • two injections one injection is an arbitrary fixed amount
  • one injection may be administered to both legs.
  • one or more can be additionally administered.
  • treatment with a growth factor mixture or platelet-rich plasma of the present disclosure can also be used to treat joints, including knees, e.g., knee osteoarthritis, hip osteoarthritis, meniscus It can be used to treat injuries, ligament injuries, knee joint pain, and the like.
  • the growth factor mixture or platelet-rich plasma of the present disclosure is used in body tissues such as skin, bone, ligaments, Achilles tendon, tenosynovitis, alveolar bone, meniscus, ovary, endometrium, cornea, and other soft tissues. It can also be used for regenerative therapy.
  • the growth factor mixture or platelet-rich plasma of the present disclosure can be used to promote wound healing (early healing), relieve pain, assist healing of surgical wounds, and the like.
  • the growth factor mixture or platelet-rich plasma of the present disclosure can also be used for cosmetic purposes such as hair thinning treatment. can be adjusted.
  • the growth factor mixture or platelet-rich plasma of the present disclosure is administered for cosmetic purposes, for example, one dose every other week, or two doses every month, repeated 2-3 times. can also
  • Short Protocols in Molecular Biology A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F.; M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M.; A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F.; M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; J. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, Supplementary Volume Experimental Medicine "Gene Introduction & Expression Analysis Experimental Method” Yodosha, 1997, etc., and these are the relevant parts (may be all) of this specification. is incorporated by reference.
  • FIG. 1 shows a series of steps from blood collection to production of the growth factor mixture.
  • blood was collected (four blood collection tubes/about 40 mL) at a clinic.
  • four heparin blood collection tubes (Venoject II vacuum blood collection tube (sterilized), containing heparin sodium) were used.
  • BD Vacutainer CPT can also be used in place of the heparin blood collection tube.
  • the mixture was gently inverted about 10 times and transported under refrigeration. At this time, the blood can also be pre-chilled at 0-10° C. for 0-72 hours.
  • the blood was returned to room temperature as soon as it arrived at the cell processing facility.
  • Four heparin blood collection tubes were centrifuged using a tabletop multi-tube centrifuge H-40F (swing rotor: RF-121-S/manufactured by Kokusan Co., Ltd.) (first centrifugation: 940 G, 15 minutes, 20-25°C).
  • first centrifugation 1500G, 15 minutes.
  • the supernatant (plasma + leukocyte layer) was collected in a 50 mL centrifuge tube (manufactured by Corning) to obtain W-PRP (WBC-containing platelet-rich plasma; leukocyte-containing platelet-rich plasma).
  • W-PRP WBC-containing platelet-rich plasma; leukocyte-containing platelet-rich plasma.
  • This leukocyte-containing platelet-rich plasma was subjected to the second centrifugation (940 G, 15 minutes, 20 to 25°C) using a tabletop multiple rack centrifuge H-40F (swing rotor: RF-121-S/manufactured by Kokusan Co., Ltd.). did When using BD Vacutainer CPT, second centrifugation: 1500 G, 15 minutes.
  • the supernatant was removed (precipitate + supernatant 0.6 mL or less), and PBS (-) was added so that the total volume was 3.6 mL.
  • An additional 0.9 mL of 2% CaCl 2 was added (Nipro Calcium Chloride Injection 2% "NP"). The mixture was well stirred to activate platelets and release growth factors (20 min, 20-25° C.).
  • a 5 mL or 10 mL syringe was filled with the activated leukocyte-containing platelet-rich plasma solution, and acellular filtering was performed.
  • a cell-free filter Minisart pore size 0.45 ⁇ m (manufactured by Sartorius) was used.
  • FIG. 2 shows a series of steps from blood collection to production of the growth factor mixture.
  • blood was collected (three blood collection tubes/about 30 mL) at a clinic.
  • three heparin blood collection tubes (BD Vacutainer blood collection tube, containing heparin sodium) were used.
  • BD Vacutainer CPT can also be used in place of the heparin blood collection tube.
  • the mixture was gently inverted about 20 times and transported under refrigeration. At this time, the blood can also be pre-chilled at 0-15° C. for 0-72 hours.
  • Blood was brought to room temperature upon arrival at the cell processing facility. Transfer 30 mL of blood from three heparin collection tubes to one lymphocyte separation tube containing lymphocyte separation solution (Leucosep (trademark) blood volume 30 mL, manufactured by Greiner Japan), and centrifuge LCX-100 (Tomy Seiko Co., Ltd.) was used to centrifuge one lymphocyte separation tube (first centrifugation: 1200 G, 15 min, 15 to 25° C.). If BD Vacutainer CPT is used, centrifugation (first centrifugation: 1500 G, 15 minutes) may be performed without transferring to a lymphocyte separation tube.
  • lymphocyte separation solution Leucosep (trademark) blood volume 30 mL, manufactured by Greiner Japan
  • centrifuge LCX-100 Tomy Seiko Co., Ltd.
  • -PRP WBC-containing platelet-rich plasma; leukocyte-containing platelet-rich plasma
  • This leukocyte-containing platelet-rich plasma was subjected to a second centrifugation (1200 G, 15 min, 15-25°C) using a tabletop multi-centrifuge LCX-100 (manufactured by Tomy Seiko) (BD Vacutainer CPT was used. second centrifugation: 1500 G, 15 min).
  • the supernatant was removed (precipitate + supernatant 1.0 mL or less), and PBS (-) was added so that the total volume was 6 mL.
  • a 10 mL syringe was filled with the activated leukocyte-containing platelet-rich plasma solution, and acellular filtering was performed.
  • a cell-free filter Minisart pore size 0.2 ⁇ m (manufactured by Sartorius) was used.
  • Example 3 Analysis of growth factor mixture
  • the amount and concentration of growth factors in the growth factor mixture prepared as in Example 1 or 2 are analyzed.
  • Analysis of major growth factors using enzyme-linked immunosorbent assay (ELISA) reveals increased amounts and concentrations of platelet-derived growth factors such as PDGF, TGF- ⁇ , VEGF, EGF, and FGF.
  • ELISA enzyme-linked immunosorbent assay
  • Example 4 Preparation of growth factor mixture (3)
  • the sequence from blood collection to production of growth factor mixture is the same as in Figure 1.
  • blood was collected (four blood collection tubes/about 40 mL) at a clinic.
  • four heparin blood collection tubes (Venoject II vacuum blood collection tube (sterilized), containing heparin sodium) were used.
  • BD Vacutainer CPT can also be used in place of the heparin blood collection tube.
  • the mixture was gently inverted about 10 times and transported under refrigeration. At this time, the blood can also be pre-chilled at 0-10° C. for 0-72 hours.
  • the blood was returned to room temperature as soon as it arrived at the cell processing facility.
  • Four heparin blood collection tubes were centrifuged using a tabletop multiple centrifuge H-40F (swing rotor: RF-121-S/manufactured by Kokusan Co., Ltd.) (first centrifugation: 940 G, 15 min, 20-25° C.).
  • first centrifugation 940 G, 15 min, 20-25° C.
  • first centrifugation 1500G, 15 minutes.
  • the supernatant was collected in a 50 mL centrifuge tube (manufactured by Corning) to obtain platelet-rich plasma.
  • This platelet-rich plasma was subjected to a second centrifugation (940 G, 15 min, 20-25° C.) using a tabletop multiple rack centrifuge H-40F (swing rotor: RF-121-S/manufactured by Kokusan Co., Ltd.). .
  • second centrifugation 1500 G, 15 minutes.
  • the supernatant was removed (precipitate + supernatant 0.6 mL or less), and PBS (-) was added so that the total volume was 3.6 mL.
  • An additional 0.9 mL of 2% CaCl 2 was added (Nipro Calcium Chloride Injection 2% "NP"). The mixture was well stirred to activate platelets and release growth factors (20 min, 20-25° C.).
  • a 5 mL or 10 mL syringe was filled with the activated platelet-rich plasma solution, and acellular filtering was performed.
  • a cell-free filter Minisart pore size 0.45 ⁇ m (manufactured by Sartorius) was used.
  • Example 5 Preparation of growth factor mixture (4)
  • blood was collected (three blood collection tubes/about 30 mL) at a clinic.
  • three heparin blood collection tubes (BD Vacutainer blood collection tube, containing heparin sodium) were used.
  • BD Vacutainer CPT can also be used in place of the heparin blood collection tube.
  • the mixture was gently inverted about 20 times and transported under refrigeration.
  • the blood can also be pre-chilled at 0-15° C. for 0-72 hours.
  • Blood was brought to room temperature upon arrival at the cell processing facility. Transfer 30 mL of blood from three heparin collection tubes to one lymphocyte separation tube containing lymphocyte separation solution (Leucosep (trademark) blood volume 30 mL, manufactured by Greiner Japan), and centrifuge LCX-100 (Tomy Seiko Co., Ltd.) was used to centrifuge one lymphocyte separation tube (first centrifugation: 1200 G, 15 min, 15 to 25° C.). If BD Vacutainer CPT is used, centrifugation (first centrifugation: 1500 G, 15 minutes) may be performed without transferring to a lymphocyte separation tube.
  • lymphocyte separation solution Leucosep (trademark) blood volume 30 mL, manufactured by Greiner Japan
  • centrifuge LCX-100 Tomy Seiko Co., Ltd.
  • the supernatant was removed (precipitate + supernatant 1.0 mL or less), and PBS (-) was added so that the total volume was 6 mL.
  • a 10 mL syringe was filled with the activated platelet-rich plasma solution, and acellular filtering was performed.
  • a cell-free filter Minisart pore size 0.2 ⁇ m (manufactured by Sartorius) was used.
  • Example 6 Preparation of growth factor mixture (5)
  • blood was collected (four blood collection tubes/about 40 mL) at a clinic.
  • four heparin blood collection tubes (Venoject II vacuum blood collection tube (sterilized), containing heparin sodium) were used.
  • BD Vacutainer CPT can also be used in place of the heparin blood collection tube.
  • the mixture was gently inverted about 10 times and transported under refrigeration. At this time, the blood can also be pre-chilled at 0-10° C. for 0-72 hours.
  • the blood was returned to room temperature as soon as it arrived at the cell processing facility.
  • Four heparin blood collection tubes were centrifuged using a tabletop multi-tube centrifuge H-40F (swing rotor: RF-121-S/manufactured by Kokusan Co., Ltd.) (first centrifugation: 940 G, 15 minutes, 20-25°C).
  • first centrifugation 940 G, 15 minutes, 20-25°C.
  • centrifugation 1500 G, 15 minutes (heparin) or 20 minutes (citric acid), centrifugation at 20-25°C.
  • the centrifuge may be of another model, and the rotor may be of the swing type or of the angle type.
  • the supernatant was collected in a 50 mL centrifuge tube (manufactured by Corning) to obtain platelet-rich plasma.
  • a centrifuge tube manufactured by Corning
  • PBS - was added to this platelet-rich plasma so that the total volume was 30 mL or more, and then a desktop multi-centrifuge centrifuge H-40F (swing rotor: RF-121-S/manufactured by Kokusan Co., Ltd.) was used.
  • a second centrifugation (940 G, 15 min, 20-25° C.) was performed.
  • 2nd centrifugation 940-1800 G, 15 minutes, 20-25° C. when transferred to a lymphocyte separation tube containing a lymphocyte separation solution in the first centrifugation and centrifuged.
  • 2nd spin 1500 G-1800 G, 15-20 min if BD Vacutainer CPT is used in 1st spin. Cells other than platelets may be removed with a filter or the like before centrifugation.
  • the supernatant was removed (precipitate + 1 mL or less of supernatant), and PBS (-) was added so that the total volume was 4.8 mL.
  • An additional 1.2 mL of 2% CaCl 2 was added (Nipro Calcium Chloride Injection 2% "NP").
  • the concentration and liquid volume of the total volume and CaCl 2 may be changed. For example, if the liquid volume is 1.2 mL or less when the supernatant is removed, the total volume is PBS(-) may be added to make 4 mL, and 1 mL of 2% CaCl 2 may be added. Then, the mixture was stirred well to activate platelets and release growth factors (30 minutes, 20-25°C). The activation time can be adjusted as appropriate, and may be within 30 minutes or extended up to 120 minutes.
  • the activated platelet-rich plasma solution was frozen (30 minutes or more, -80°C) and thawed.
  • Cells other than platelets may be removed by a filter or the like before freezing.
  • Thawing may be carried out on the next day or later, and the temperature and time for thawing may be room temperature for 30 minutes or longer or 4°C for 2 hours or longer.
  • centrifugation 14000 rpm, 5 minutes, 20-25° C.
  • Minisart pore size 0.45 ⁇ m (manufactured by Sartorius) was used.
  • Example 7 Measurement of PDGF-BB and VEGF
  • a series of flows from blood collection to production of the growth factor mixture are the same as in FIG.
  • blood was collected (four blood collection tubes/about 40 mL) at a clinic.
  • four heparin blood collection tubes (Venoject II vacuum blood collection tube (sterilized), containing heparin sodium) were used.
  • the mixture was gently inverted about 10 times and transported under refrigeration.
  • the blood can also be pre-chilled at 0-10° C. for 0-72 hours.
  • the blood was returned to room temperature as soon as it arrived at the cell processing facility.
  • Four heparin blood collection tubes were centrifuged using a tabletop multi-tube centrifuge H-40F (swing rotor: RF-121-S/manufactured by Kokusan Co., Ltd.) (first centrifugation: 940 G, 15 minutes, 20-25°C).
  • the supernatant (plasma + leukocyte layer) of one of the four blood collection tubes was used as PRP.
  • the re-centrifuged supernatant was used, subjected to decellularization filtering, and then frozen and stored until measurement.
  • the remaining three supernatants (plasma + leukocyte layer) were collected in 15 mL centrifuge tubes (manufactured by Corning) to obtain W-PRP (WBC-containing platelet-rich plasma; leukocyte-containing platelet-rich plasma).
  • This leukocyte-containing platelet-rich plasma was subjected to the second centrifugation (940 G, 15 minutes, 20 to 25°C) using a tabletop multiple rack centrifuge H-40F (swing rotor: RF-121-S/manufactured by Kokusan Co., Ltd.). did
  • a 5 mL or 10 mL syringe was filled with the activated leukocyte-containing platelet-rich plasma solution, and acellular filtering was performed.
  • a cell-free filter Minisart pore size 0.45 ⁇ m (manufactured by Sartorius) was used.
  • PDGF-BB and VEGF were measured by ELISA and compared with conventional platelet-rich plasma (PRP). The results are shown in Tables 1 and 2 below.
  • growth factors can be efficiently recovered from platelet-rich plasma and activated, so a wide range of applications can be expected in the medical field, including regenerative medicine.

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US20150150942A1 (en) * 2007-10-15 2015-06-04 The Regents Of The University Of Colorado, A Body Corporate Methods for extracting platelets and compositions obtained therefrom
US20150224173A1 (en) * 2012-08-17 2015-08-13 Kasiak Research Pvt. Ltd. Method of preparing a growth factor concentrate derived from human platelets
JP2016529283A (ja) * 2013-08-27 2016-09-23 クック・ジェネラル・バイオテクノロジー・エルエルシー 濃厚血小板から誘導可能な生理活性組成物並びにその調製方法及び使用方法
WO2019187200A1 (ja) * 2018-03-30 2019-10-03 セルソース株式会社 成長因子混合物およびその調製方法
WO2021009660A1 (en) * 2019-07-12 2021-01-21 Vasanthi Palanivel Composition and methods for improving thickness and receptivity of endometrial lining

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US20220127573A1 (en) * 2019-02-15 2022-04-28 Terumo Kabushiki Kaisha Platelet lysate production method, production system, and bag set

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150150942A1 (en) * 2007-10-15 2015-06-04 The Regents Of The University Of Colorado, A Body Corporate Methods for extracting platelets and compositions obtained therefrom
US20150224173A1 (en) * 2012-08-17 2015-08-13 Kasiak Research Pvt. Ltd. Method of preparing a growth factor concentrate derived from human platelets
JP2016529283A (ja) * 2013-08-27 2016-09-23 クック・ジェネラル・バイオテクノロジー・エルエルシー 濃厚血小板から誘導可能な生理活性組成物並びにその調製方法及び使用方法
WO2019187200A1 (ja) * 2018-03-30 2019-10-03 セルソース株式会社 成長因子混合物およびその調製方法
WO2021009660A1 (en) * 2019-07-12 2021-01-21 Vasanthi Palanivel Composition and methods for improving thickness and receptivity of endometrial lining

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