WO2022239870A1 - 血液由来成長因子含有組成物及びその調製方法 - Google Patents
血液由来成長因子含有組成物及びその調製方法 Download PDFInfo
- Publication number
- WO2022239870A1 WO2022239870A1 PCT/JP2022/020281 JP2022020281W WO2022239870A1 WO 2022239870 A1 WO2022239870 A1 WO 2022239870A1 JP 2022020281 W JP2022020281 W JP 2022020281W WO 2022239870 A1 WO2022239870 A1 WO 2022239870A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- blood
- platelet
- rich plasma
- growth factor
- minutes
- Prior art date
Links
- 239000008280 blood Substances 0.000 title claims abstract description 121
- 210000004369 blood Anatomy 0.000 title claims abstract description 120
- 239000003102 growth factor Substances 0.000 title claims abstract description 99
- 239000000203 mixture Substances 0.000 title claims abstract description 74
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 238000000034 method Methods 0.000 claims abstract description 71
- 239000003146 anticoagulant agent Substances 0.000 claims abstract description 26
- 229940127219 anticoagulant drug Drugs 0.000 claims abstract description 26
- 238000010257 thawing Methods 0.000 claims abstract description 15
- 229910021645 metal ion Inorganic materials 0.000 claims abstract description 10
- 230000003213 activating effect Effects 0.000 claims abstract description 7
- 210000004623 platelet-rich plasma Anatomy 0.000 claims description 80
- 238000005119 centrifugation Methods 0.000 claims description 31
- 238000007710 freezing Methods 0.000 claims description 22
- 230000008014 freezing Effects 0.000 claims description 22
- 238000001914 filtration Methods 0.000 claims description 13
- 238000004108 freeze drying Methods 0.000 claims description 12
- 210000002381 plasma Anatomy 0.000 claims description 12
- 239000011148 porous material Substances 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 11
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 9
- 239000008188 pellet Substances 0.000 claims description 8
- 150000004697 chelate complex Chemical class 0.000 claims description 7
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 3
- 241000124008 Mammalia Species 0.000 abstract description 8
- 230000029663 wound healing Effects 0.000 abstract description 7
- 230000017423 tissue regeneration Effects 0.000 abstract description 5
- 239000013522 chelant Substances 0.000 abstract description 3
- 102000004127 Cytokines Human genes 0.000 description 37
- 108090000695 Cytokines Proteins 0.000 description 37
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 27
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 27
- 101000599048 Homo sapiens Interleukin-6 receptor subunit alpha Proteins 0.000 description 21
- 102100037792 Interleukin-6 receptor subunit alpha Human genes 0.000 description 21
- 230000003110 anti-inflammatory effect Effects 0.000 description 21
- 238000000926 separation method Methods 0.000 description 16
- 230000002757 inflammatory effect Effects 0.000 description 15
- 239000000243 solution Substances 0.000 description 10
- 239000012528 membrane Substances 0.000 description 8
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 7
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 7
- 210000000601 blood cell Anatomy 0.000 description 7
- 238000001035 drying Methods 0.000 description 7
- 239000002904 solvent Substances 0.000 description 7
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 102000004889 Interleukin-6 Human genes 0.000 description 6
- 108090001005 Interleukin-6 Proteins 0.000 description 6
- 238000009777 vacuum freeze-drying Methods 0.000 description 6
- 230000001684 chronic effect Effects 0.000 description 5
- 238000005057 refrigeration Methods 0.000 description 5
- 102000003745 Hepatocyte Growth Factor Human genes 0.000 description 4
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 description 4
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 4
- 102000014429 Insulin-like growth factor Human genes 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 210000003743 erythrocyte Anatomy 0.000 description 4
- 238000002844 melting Methods 0.000 description 4
- 230000008018 melting Effects 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 241000282414 Homo sapiens Species 0.000 description 3
- 206010061218 Inflammation Diseases 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 229910001424 calcium ion Inorganic materials 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 230000004054 inflammatory process Effects 0.000 description 3
- 210000000265 leukocyte Anatomy 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 239000008215 water for injection Substances 0.000 description 3
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 2
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 2
- 208000000491 Tendinopathy Diseases 0.000 description 2
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 2
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 235000011148 calcium chloride Nutrition 0.000 description 2
- BPKIGYQJPYCAOW-FFJTTWKXSA-I calcium;potassium;disodium;(2s)-2-hydroxypropanoate;dichloride;dihydroxide;hydrate Chemical compound O.[OH-].[OH-].[Na+].[Na+].[Cl-].[Cl-].[K+].[Ca+2].C[C@H](O)C([O-])=O BPKIGYQJPYCAOW-FFJTTWKXSA-I 0.000 description 2
- 238000012790 confirmation Methods 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- QLBHNVFOQLIYTH-UHFFFAOYSA-L dipotassium;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [K+].[K+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O QLBHNVFOQLIYTH-UHFFFAOYSA-L 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000000703 high-speed centrifugation Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000001172 regenerating effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- LOGFVTREOLYCPF-KXNHARMFSA-N (2s,3r)-2-[[(2r)-1-[(2s)-2,6-diaminohexanoyl]pyrrolidine-2-carbonyl]amino]-3-hydroxybutanoic acid Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]1CCCN1C(=O)[C@@H](N)CCCCN LOGFVTREOLYCPF-KXNHARMFSA-N 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 201000004624 Dermatitis Diseases 0.000 description 1
- 206010012438 Dermatitis atopic Diseases 0.000 description 1
- 201000009273 Endometriosis Diseases 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 238000012424 Freeze-thaw process Methods 0.000 description 1
- 101001076407 Homo sapiens Interleukin-1 receptor antagonist protein Proteins 0.000 description 1
- -1 IL-1β Proteins 0.000 description 1
- 229940119178 Interleukin 1 receptor antagonist Drugs 0.000 description 1
- 102000003777 Interleukin-1 beta Human genes 0.000 description 1
- 108090000193 Interleukin-1 beta Proteins 0.000 description 1
- 102000051628 Interleukin-1 receptor antagonist Human genes 0.000 description 1
- 206010028331 Muscle rupture Diseases 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 208000023835 Tendon disease Diseases 0.000 description 1
- 206010043255 Tendonitis Diseases 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 230000002491 angiogenic effect Effects 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 201000008937 atopic dermatitis Diseases 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 235000010216 calcium carbonate Nutrition 0.000 description 1
- MKJXYGKVIBWPFZ-UHFFFAOYSA-L calcium lactate Chemical compound [Ca+2].CC(O)C([O-])=O.CC(O)C([O-])=O MKJXYGKVIBWPFZ-UHFFFAOYSA-L 0.000 description 1
- 239000001527 calcium lactate Substances 0.000 description 1
- 229960002401 calcium lactate Drugs 0.000 description 1
- 235000011086 calcium lactate Nutrition 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000019262 disodium citrate Nutrition 0.000 description 1
- 239000002526 disodium citrate Substances 0.000 description 1
- CEYULKASIQJZGP-UHFFFAOYSA-L disodium;2-(carboxymethyl)-2-hydroxybutanedioate Chemical compound [Na+].[Na+].[O-]C(=O)CC(O)(C(=O)O)CC([O-])=O CEYULKASIQJZGP-UHFFFAOYSA-L 0.000 description 1
- 210000004696 endometrium Anatomy 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000003407 interleukin 1 receptor blocking agent Substances 0.000 description 1
- 229940100601 interleukin-6 Drugs 0.000 description 1
- 238000000464 low-speed centrifugation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000010309 melting process Methods 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 230000003239 periodontal effect Effects 0.000 description 1
- 230000010118 platelet activation Effects 0.000 description 1
- 229920005646 polycarboxylate Polymers 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 201000004415 tendinitis Diseases 0.000 description 1
- 208000013515 tendinosis Diseases 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000003813 thin hair Effects 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
Images
Classifications
-
- 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
-
- 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
- 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
- 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
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/20—Interleukins [IL]
-
- 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 invention relates to a blood-derived growth factor-containing composition and a preparation method thereof.
- platelet-rich plasma platelet rich plasma: hereinafter also referred to as "PRP"
- PRP platelet rich plasma
- This platelet-rich plasma is rich in many growth factors. Since these growth factors play an effective role in wound healing and tissue regeneration, platelet-rich plasma is a promising material in the field of regenerative medicine (eg, Patent Document 1).
- the preparation of PRP includes the process of activating platelets and releasing growth factors contained in platelets.
- Platelet-derived factor release stimulators such as thrombin and calcium chloride have been used for platelet activation.
- the main object of the present invention is to provide a blood-derived growth factor-containing composition effective for wound healing and tissue regeneration and a method for preparing the blood-derived growth factor-containing composition. Furthermore, another object of the present invention is to provide a blood-derived growth factor-containing composition capable of relieving severe pain upon administration and a method for preparing the blood-derived growth factor-containing composition.
- VEGF vascular endothelial growth factor
- the present inventors have developed a blood-derived growth factor-containing composition capable of relieving severe pain during administration by performing filtration treatment with a filter of a predetermined size. We have found that it is possible to prepare Specifically, it is the following invention.
- a method for preparing a blood-derived growth factor-containing composition from mammalian blood comprising the steps of: adding to the blood an anticoagulant capable of forming a chelate complex with a metal ion; separating platelet-rich plasma containing buffy coat components from the blood; The blood to which is added is subjected to a first centrifugation treatment at 5 to 2000 G for 1 to 60 minutes to recover an upper layer containing plasma and buffy coat, and in the step of recovering the upper layer , a method in which the volume ratio of the upper layer portion to be recovered is 1:0.1 to 0.5 based on the volume of the total liquid volume; [2] After the step of collecting the upper layer, further subjecting the upper layer to a second centrifugation treatment at 100 to 2500 G for 1 to 30 minutes to pellet platelets and buffy coat components; removing the supernatant and suspending the platelets and the buffy coat component to obtain a platelet-rich plasma; and The method according to [1]
- the step of activating the platelet-rich plasma by freezing and thawing comprises freezing by treatment at ⁇ 200° C. to ⁇ 20° C. for 10 minutes or more, followed by thawing by treatment at 20° C. to 50° C. for 10 minutes or more.
- a method for preparing a blood-derived growth factor-containing composition from mammalian blood comprising the steps of: adding to the blood an anticoagulant capable of forming a chelate complex with metal ions; a step of separating platelet-rich plasma containing a buffy coat component from the blood, a step of freezing and thawing the platelet-rich plasma to activate it, and a filter having a pore size of 0.2 to 1 ⁇ m, the activated filtering the platelet-rich plasma; [10] A blood-derived growth factor-containing composition prepared by the method according to any one of [1] to [7] and [9] above; [11
- a blood-derived growth factor-containing composition effective for wound healing and tissue regeneration and a method for preparing the blood-derived growth factor-containing composition. Furthermore, according to the present invention, it is possible to provide a blood-derived growth factor-containing composition capable of alleviating severe pain during administration and a method for preparing the blood-derived growth factor-containing composition.
- FIG. 1 is a graph showing the amounts of growth factor (VEGF) and anti-inflammatory cytokine (IL-1Ra) in blood-derived growth factor-containing compositions of Examples 6-10.
- FIG. 2 is a graph showing the amount of growth factor (VEGF) in a blood-derived growth factor-containing composition obtained under predetermined separation conditions.
- FIG. 3 is a graph showing the amount of anti-inflammatory cytokine (IL-1Ra) in blood-derived growth factor-containing compositions obtained under predetermined separation conditions.
- a method for preparing the blood-derived growth factor-containing composition according to the present embodiment will be described below, and then the blood-derived growth factor-containing composition according to the present embodiment obtained by the above-described preparation method will be described.
- a method for preparing a blood-derived growth factor-containing composition is a method for preparing a blood-derived growth factor composition from mammalian blood, wherein an anticoagulant capable of forming a chelate complex with a metal ion is added to the blood. and a step of separating platelet-rich plasma containing a buffy coat component from the blood to which the anticoagulant has been added (hereinafter also referred to as a “separation step”).
- mammal blood is not particularly limited as long as it contains a growth factor, and is, for example, blood derived from humans, pigs, cows, horses, dogs, cats and monkeys, preferably human is the blood of Also, said "mammalian blood” is preferably autologous.
- ⁇ Addition process> blood to which an anticoagulant that forms a chelate complex with metal ions has been added is used.
- the anticoagulant may be added to the collected blood later, but it is desirable to collect the blood in advance in a container such as a blood collection tube or blood collection bag containing the anticoagulant.
- Anticoagulants that form chelate complexes with metal ions include compounds that form chelate complexes with metal ions (such as calcium ions), such as polycarboxylic acids and salts thereof.
- polycarboxylic acids include citric acid and EDTA.
- Polycarboxylates include EDTA salts such as 2Na citrate (citrate), EDTA-2K, and EDTA-2Na.
- the anticoagulant may be added as a solid, or may be added as a solution in which the anticoagulant is dissolved in a solvent such as pure water (preferably a physiologically acceptable one).
- the final concentration of anticoagulant in whole blood after addition of anticoagulant is preferably 0.5 in the case of disodium citrate. to 1.5% by mass, more preferably 0.8 to 1.0% by mass, and in the case of EDTA-2K, preferably 0.5 to 2.0 mg/mL, more preferably 1.1 to 1 .7 mg/mL, and in the case of EDTA-2Na, preferably 0.5-2.0 mg/mL, more preferably 1.0-1.5 mg/mL.
- the method of the present embodiment further includes the step of pre-chilling the anticoagulant-added blood before the step of separating platelet-rich plasma from the blood to which the anticoagulant that forms a chelate complex with the metal ion is added.
- the refrigeration temperature in the step of pre-chilling the blood is, for example, 0-10°C.
- the process time of the step of pre-chilling the blood is, for example, more than 0 hours and 72 hours or less.
- the lower limit of the process time may be 30 minutes or more, 1 hour or more, 2 hours or more, 4 hours or more, or 8 hours or more.
- the upper limit of the process time may be 60 hours or less, 48 hours or less, 36 hours or less, or 24 hours or less.
- the method of this embodiment includes the step of separating platelet-rich plasma containing buffy coat components from blood to which an anticoagulant has been added.
- buffy coat refers to a thin white layer containing a large number of leukocytes formed between erythrocytes and plasma fractions when blood is centrifuged.
- “comprising a buffy coat component” means containing at least part of the buffy coat.
- the temperature of blood and platelet-rich plasma is preferably controlled between 4 and 25°C during this separation step.
- the blood is usually subjected to a first centrifugation process at a relatively low speed to separate the red blood cell-containing fraction, the buffy coat, the platelet-rich plasma (PRP), and the platelet-poor plasma.
- platelet-rich plasma is obtained.
- the separation step preferably includes a first relatively low speed centrifugation process and a second relatively high speed centrifugation process.
- the first centrifugation process and the second centrifugation process may each be independently performed multiple times.
- the platelet-rich plasma and buffy coat may be obtained by a relatively slow first centrifugation process alone.
- the first centrifugation treatment, the second centrifugation treatment, and the concentration/purification treatment of platelets and buffy coat components are described in detail below.
- the relatively low-speed first centrifugation treatment is preferably centrifugation at 5 to 2000 G (corresponding to about 200 to 2000 rpm in a normal centrifuge). Rotation conditions may be 100-1000G, 150-750G, and 200-500G.
- the treatment time may be 1 to 60 minutes, 1 to 30 minutes, 5 to 20 minutes, 10 to 15 minutes.
- the volume ratio of the upper layer portion to be recovered is preferably 1:0.1 to 0.5, more preferably 1:0.2 to 0.5, based on the volume of the total liquid volume. 5, more preferably 1:0.3-0.5, particularly preferably 1:0.35-0.5, most preferably 1:0.4-0.5.
- the relatively high-speed second centrifugation process is preferably centrifugation at 100-2500 G (equivalent to about 700-5000 rpm in a normal centrifuge).
- Rotation conditions may be 1100-2000G, 1200-1500G.
- the treatment time may be 1-30 minutes, 5-20 minutes, or 10-15 minutes.
- the platelets and buffy coat components can be purified and concentrated.
- any volume can be removed in the step of removing the supernatant.
- the volume ratio is preferably 1: 1 to 10, more preferably 1: 1 to 5, and still more preferably 1: 1 to 3. The supernatant can be retained and the rest can be removed.
- Addition of drugs is known as a method of activating platelet-rich plasma.
- Agents that activate platelet-rich plasma generally include calcium ion source compounds such as calcium chloride, calcium phosphate, calcium lactate, and calcium carbonate.
- activation of platelet-rich plasma can be achieved by freezing and thawing without adding drugs. Therefore, the use of calcium ion source compounds can be minimized and additives can be minimized.
- Freezing of platelet-rich plasma can be performed by exposing the platelet-rich plasma to a low temperature at which freezing occurs. Specifically, the platelet-rich plasma is placed under temperature conditions of, for example, ⁇ 10° C. or less, ⁇ 20° C. or less, ⁇ 100° C. or less, or ⁇ 200° C. or less, preferably ⁇ 20° C. to ⁇ 200° C. Can be frozen. This may be done by contact with liquid nitrogen or storage in a freezer.
- the freezing process time may be 1 minute or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 25 minutes or more, or 30 minutes or more. On the other hand, it may be 180 minutes or less, 150 minutes or less, 90 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, or 30 minutes or less.
- freezing can be performed, for example, by immersing a container containing platelet-rich plasma in liquid nitrogen for 1 minute or longer.
- Melting of platelet-rich plasma can be performed by exposing the platelet-rich plasma to a temperature at which melting occurs. Specifically, frozen platelet-rich plasma is treated at a temperature of, for example, 20°C or higher, 25°C or higher, or 30°C or higher and 50°C or lower, 40°C or lower, or 38°C or lower, preferably 20°C to 50°C. Melting can be achieved by subjecting to conditions.
- the time for the step of thawing platelet-rich plasma and activating platelets is not particularly limited. The lower limit of the melting step time may be 1 minute or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 25 minutes or more, or 30 minutes or more.
- the upper limit of the melting process time may be 180 minutes or less, 150 minutes or less, 90 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, or 30 minutes or less.
- platelets can be activated by allowing a container containing platelet-rich plasma to stand at room temperature for 10 to 30 minutes.
- a solvent may be added to the activated platelet-rich plasma to resuspend.
- the solvent for resuspension is any physiologically acceptable solvent. Examples thereof include plasma, physiological saline, buffered physiological saline ⁇ eg, phosphate-buffered saline (PBS) ⁇ , Ringer's solution (eg, lactated Ringer's solution, acetated Ringer's solution, bicarbonate Ringer's solution), and the like.
- PBS phosphate-buffered saline
- Ringer's solution eg, lactated Ringer's solution, acetated Ringer's solution, bicarbonate Ringer's solution
- Any platelet resuspension resuspended in any solvent is referred to herein as "platelet-rich plasma”.
- the above solvent may be further added to this resuspension, and the liquid after addition is also referred to as "platelet-rich plasma.”
- the method of this embodiment may further comprise removing blood cells from the platelet-rich plasma.
- the platelet-rich plasma is preferably controlled at 4-25°C during this cell removal step.
- Plate-rich plasma contains platelets.” Moreover, according to the method of this embodiment, the platelet-rich plasma also contains white blood cells derived from the buffy coat. Additionally, the platelet-rich plasma may contain a small amount of red blood cells.
- this step is preferably a filtration treatment step for removing blood cells from the platelet-rich plasma by filtration treatment.
- the filters used for filtering there are no restrictions on the filters used for filtering, as long as they can remove blood cells. Since platelets are the smallest in blood cell size at about 2 ⁇ m, for example, membrane filters with pore sizes of 1 ⁇ m or less, 0.7 ⁇ m or less, or 0.5 ⁇ m or less can be used. There is no lower limit to the pore size of the membrane filter, but excessively small pore sizes increase the risk of clogging. Therefore, the pore diameter is preferably 0.2 ⁇ m or more, 0.3 ⁇ m or more, or 0.4 ⁇ m or more. Membrane filters commonly available on the market that satisfy this range include a membrane filter with a pore size of about 0.2 ⁇ m and a membrane filter with a pore size of about 0.45 ⁇ m. Any of them can be used in this embodiment, and a membrane filter with a pore size of about 0.45 ⁇ m is the most suitable membrane filter.
- the filtering process may be performed by centrifugation using a centrifuge tube provided with the aforementioned filter.
- the centrifugation at this time is, for example, centrifugation at 800 to 1800 G (corresponding to about 1500 to 4000 rpm in a normal centrifuge).
- the process time is, for example, 1 to 30 minutes, 5 to 15 minutes, or 10 minutes.
- the method of this embodiment may further comprise freeze-drying the platelet-rich plasma.
- the freeze-drying method is not particularly limited as long as it is a conventional freeze-drying method for proteins.
- platelet-rich plasma is frozen and then vacuum freeze-dried.
- the temperature of the platelet-rich plasma or the environmental temperature is adjusted to -60°C or lower, -70°C or lower, or -80°C or lower. This may be done by contact with liquid nitrogen or storage in a freezer.
- the execution time of the freezing process can be instant.
- the freezing process is carried out at the above temperatures for 6-24 hours to ensure that all the platelet-rich plasma is frozen. That is, the interval from the start time of the freezing treatment to the start time of the vacuum freeze-drying treatment may be 0 to 24 hours or 6 to 24 hours.
- the temperature conditions for the vacuum freeze-drying process There are no particular restrictions on the temperature conditions for the vacuum freeze-drying process.
- the upper limit of the temperature conditions may be ⁇ 30° C. or less, ⁇ 35° C. or less, ⁇ 40° C. or less, or ⁇ 45° C. or less.
- the lower limit is not particularly limited, it may be -80°C or higher, -75°C or higher, -70°C or higher, -65°C or higher, -55°C or higher, or -50°C or higher.
- the upper limit of the atmospheric pressure condition may be 20 Pa or less, 18 Pa or less, 16 Pa or less, or 15 Pa or less.
- the lower limit is 0 Pa or more, and may be 1 Pa or more, 2 Pa or more, 3 Pa or more, 4 Pa or more, or 5 Pa or more.
- the time to perform the vacuum freeze-drying process can be set according to the amount of liquid.
- the lower limit of the freeze-drying time may be, for example, 1 hour or longer, 2 hours or longer, 3 hours or longer, 4 hours or longer, 5 hours or longer, 6 hours or longer, 7 hours or longer, and 8 hours or longer.
- the upper limit of the freeze-drying time may be 24 hours or less, 20 hours or less, 15 hours or less, 10 hours or less, or 8 hours or less.
- the blood-derived growth factor-containing composition is a blood-derived growth factor-containing composition prepared from mammalian blood and having an extremely high content of a specific growth factor. be.
- the blood-derived growth factor-containing composition may be liquid, semi-solid, or dry solid. Ingredients are described in detail below.
- the blood-derived growth factor-containing composition according to the present embodiment preferably has a growth factor VEGF content of 90 pg/mL or more, more preferably 100 pg/mL or more, still more preferably 200 pg/mL or more, particularly It is preferably 300 pg/mL or more.
- the upper limit of the VEGF content is, for example, 10000 pg/mL, although it varies depending on individual differences in mammals from which blood is collected.
- pg/mL in the specification and claims is the concentration in the platelet-rich plasma after the filtration process, or the platelet-rich plasma after the freeze-drying process is melted at 300 mg per 1 mL of solvent.
- VEGF Concentration in platelet-rich plasma.
- VEGF is a growth factor abundant in buffy coat, although it is also contained in platelets.
- VEGF has an angiogenic effect, and a composition containing a large amount of VEGF, which contains a blood-derived growth factor, can be expected to have a higher wound healing effect.
- the VEGF content (pg/1 g of dry matter) relative to the dry mass of the composition is preferably 300 pg/g or more, more preferably 700 pg/g or more, and still more preferably 1000 pg/g or more. be.
- the upper limit of the VEGF content with respect to the dry mass of the composition varies depending on the individual difference of mammals from which blood was collected, but is, for example, 30000 pg/g.
- the dry weight of the composition is the weight after drying the composition under the drying conditions described below. (Drying conditions) Using FDU-1110 (manufactured by Tokyo Rika Kikai Co., Ltd.), the composition to be dried is vacuum freeze-dried under conditions of 5 Pa and ⁇ 45° C. for 16 hours.
- the blood-derived growth factor-containing composition according to this embodiment may contain other growth factors.
- the type of growth factor is not particularly limited. TGF- ⁇ ), insulin-like growth factor (IGF), hepatocyte growth factor (HGF).
- the blood-derived growth factor-containing composition according to this embodiment may contain an anti-inflammatory cytokine: an interleukin-1 receptor antagonist (hereinafter also referred to as "IL-1Ra").
- IL-1Ra interleukin-1 receptor antagonist
- the content of IL-1Ra is preferably 100 pg/mL or more, more preferably 150 pg/mL or more, still more preferably 200 pg/mL or more.
- the upper limit of the IL-1Ra content is, for example, 50,000 pg/mL, although it varies depending on individual differences in mammals from which blood was collected.
- IL-1Ra is primarily contained in leukocytes.
- IL-1Ra acts as an antagonist of the inflammatory cytokines IL-1 ⁇ and IL-1 ⁇ and suppresses inflammation.
- the content of the anti-inflammatory cytokine IL-1Ra (pg/1 g of dry matter) relative to the dry mass of the composition is preferably 300 pg/g or more, more preferably 450 pg/g or more, and still more preferably is 600 pg/g or more.
- the upper limit of the IL-1Ra content with respect to the dry mass of the composition varies depending on individual differences in mammals from which blood was collected, but is, for example, 150000 pg/g.
- the dry weight of the composition is the weight after drying the composition under the drying conditions described above.
- the blood-derived growth factor-containing composition contains inflammatory cytokines: interleukin-1 ⁇ (hereinafter also referred to as “IL-1 ⁇ ”), interleukin-6 (hereinafter referred to as “IL -6”), and Tumor necrosis factor- ⁇ (hereinafter also referred to as “TNF- ⁇ ”).
- IL-1 ⁇ interleukin-1 ⁇
- IL-6 interleukin-6
- TNF- ⁇ Tumor necrosis factor- ⁇
- the contents of IL-1 ⁇ and IL-6 are each independently preferably 10 pg/mL or less, more preferably 9.5 pg/mL or less, still more preferably 9.0 pg/mL or less.
- the content of TNF- ⁇ is preferably 20 pg/mL or less, more preferably 19 pg/mL or less, still more preferably 18 pg/mL or less. Excessive expression of these inflammatory cytokines may lead to the development of diseases such as rheumatoid arthritis.
- the inflammatory cytokine contained in the blood-derived growth factor-containing composition according to the present embodiment has a concentration as low as the above range, such side effects are less likely to occur when administered to humans or animals.
- the contents of IL-1 ⁇ and IL-6 (pg/1 g of dry matter) relative to the dry mass of the composition are each independently preferably 30 pg/g or less, more preferably 28 pg/g or less.
- the TNF- ⁇ content (pg/1 g of dry matter) relative to the dry mass of the composition is preferably 60 pg/g or less, more preferably 57 pg/g or less, and even more preferably 54 pg/g. It is below.
- the dry weight of the composition is the weight after drying the composition under the drying conditions described above.
- the blood-derived growth factor-containing composition according to this embodiment preferably contains growth factors and anti-inflammatory cytokines in a well-balanced manner.
- the mass ratio of the anti-inflammatory cytokine IL-1Ra to the mass of the growth factor VEGF is preferably 1:1 to 50, more preferably 1:1 to 15, even more preferably. is 1:1-10.
- a blood-derived growth factor-containing composition containing a growth factor and an anti-inflammatory cytokine in such a ratio has both a wound-healing effect and an anti-inflammatory effect, and therefore exhibits a higher therapeutic effect when administered to humans and animals.
- the blood-derived growth factor-containing composition preferably contains a large amount of growth factors and little inflammatory cytokines.
- the mass ratio of the inflammatory cytokine IL-1 ⁇ to the mass of the growth factor VEGF is preferably 1:0.001-0.05, more preferably 1:0.001-0. 0.03, more preferably 1:0.001 to 0.01.
- a blood-derived growth factor-containing composition containing a growth factor and an inflammatory cytokine in such a ratio hardly causes inflammation when administered to a human body or an animal, and exhibits a high therapeutic effect.
- Blood-derived growth factor-containing compositions according to this embodiment may be in a lyophilized state.
- the blood-derived growth factor-containing composition in a freeze-dried state preferably has a growth factor VEGF content of 300 pg/g or more, more preferably 700 pg/g or more, and still more preferably 1000 pg/g. That's it.
- the upper limit of the VEGF content is, for example, 30,000 pg/g, although it varies depending on individual differences in mammals from which blood is collected.
- the blood-derived growth factor-containing composition in a lyophilized state according to this embodiment may contain other growth factors.
- the type of growth factor is not particularly limited. TGF- ⁇ ), insulin-like growth factor (IGF), hepatocyte growth factor (HGF).
- the blood-derived growth factor-containing composition in a lyophilized state may comprise the anti-inflammatory cytokine IL-1Ra.
- the content of IL-1Ra is preferably 300 pg/g or more, more preferably 450 pg/g or more, still more preferably 600 pg/g or more.
- the upper limit of the IL-1Ra content is, for example, 150,000 pg/g, although it varies depending on individual differences in mammals from which blood was collected.
- Blood-derived growth factor-containing compositions may include the inflammatory cytokines IL-1 ⁇ , IL-6 and TNF- ⁇ .
- the contents of IL-1 ⁇ and IL-6 are each independently preferably 30 pg/g or less, more preferably 28 pg/g or less, still more preferably 27 pg/g or less.
- the content of TNF- ⁇ (pg/1 g of dry matter) is preferably 60 pg/g or less, more preferably 57 pg/g or less, still more preferably 54 pg/g or less.
- the blood-derived factor-containing composition in a lyophilized state preferably contains a balance of growth factors and anti-inflammatory cytokines.
- the mass ratio of the anti-inflammatory cytokine IL-1Ra to the mass of the growth factor VEGF is preferably 1:1 to 50, more preferably 1:1 to 15, even more preferably. is 1:1-10.
- the blood-derived growth factor-containing composition preferably contains a large amount of growth factors and little inflammatory cytokines.
- the mass ratio of the inflammatory cytokine IL-1 ⁇ to the mass of the growth factor VEGF is preferably 1:0.001-0.05, more preferably 1:0.001-0. 0.03, more preferably 1:0.001 to 0.01.
- blood-derived components including buffy coat components can be easily recovered, growth factors are dramatically increased, and blood-derived components exhibit wound healing and tissue regeneration effects.
- Growth factor-containing compositions can be prepared.
- the blood-derived factor-containing composition according to this embodiment can be used for regenerative medicine and the like, particularly for PRP therapy and the like.
- one embodiment of the present invention is a method of treatment using a blood-derived growth factor-containing composition.
- the blood-derived factor-containing composition according to this embodiment can restore tissue and reduce inflammation.
- the blood-derived factor-containing composition according to the present embodiment is useful for treatment of, for example, chronic tendinosis, chronic muscle rupture (tendonitis), cartilage rupture, chronic degenerative arthritis and damaged connective tissue, atopic dermatitis and chronic wounds. treatment of chronic inflammatory skin diseases including endometriosis, improvement of implantation rate by activation of the endometrium, treatment of periodontal tissue, treatment of thinning hair, and the like.
- This plasma was subjected to a second centrifugation treatment ⁇ 2680 rpm (equivalent to 1400 G), 10 minutes, 24° C. ⁇ to separate the pellet and supernatant. After separation, the supernatant having a volume ratio of 1:2 based on the volume of the pellet was retained, and the remaining supernatant (plasma) was removed. The remaining supernatant suspended the pellet to obtain a suspension (platelet-rich plasma).
- This platelet-rich plasma was exposed to an environment of -60°C for 10 minutes and frozen, then taken out and allowed to stand in a dry bath set at 37°C for 15 minutes to melt. After thawing, 6.5 mL of lactated Ringer's solution (manufactured by Fuso Pharmaceutical Co., Ltd.) was added and resuspended to stabilize the platelet-rich plasma.
- the platelet-rich plasma was transferred to a vial, subjected to a freezing treatment at -60°C, and stored frozen at the same temperature for 24 hours.
- Table 1 shows the results.
- the contents of VEGF, IL-1Ra, IL-1 ⁇ , IL-6 and TNF- ⁇ were 100 pg/mL or higher, 100 pg/mL or higher, 10 pg/mL or lower, 10 pg/mL or lower and 20 pg/mL or lower, respectively.
- Table 2 shows the results of growth factor, anti-inflammatory cytokine, and inflammatory cytokine amount/dry matter (1 g) calculated from this.
- Table 3 shows the amount ratio of each cytokine based on the amount of the growth factor VEGF based on the results of the analysis of the samples of Examples 1 to 5.
- FIG. 1 shows the amount ratio of each cytokine when the sample prepared without pre-refrigeration after blood collection (Example 6) is taken as 100%. Pre-chilling at 4° C. for 24 hours (Example 7) or 48 hours (Example 8) increased the content of each factor to about 500% for IL-1Ra and about 200% for VEGF. On the other hand, no significant increase in each factor was observed when the product was allowed to stand at room temperature for 24 hours (Example 9) or 48 hours (Example 10) instead of pre-refrigeration.
- VEGF growth factor
- IL-1Ra anti-inflammatory cytokine
- a suspension containing no buffy coat component was also obtained by recovering the upper layer of only the plasma fraction after the first centrifugation under each separation condition except for separation condition B.
- Each obtained sample was subjected to freeze-thaw activation steps, blood cell removal steps and freeze-drying steps in the same manner as in Examples 1-5.
- Table 5 shows the anti-inflammatory cytokine (IL-1Ra) amount ratio based on the amount of the growth factor VEGF based on the above analysis results for each sample containing the buffy coat component.
- IL-1Ra anti-inflammatory cytokine
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Immunology (AREA)
- Zoology (AREA)
- Epidemiology (AREA)
- Hematology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Organic Chemistry (AREA)
- Dermatology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Materials For Medical Uses (AREA)
Abstract
Description
〔1〕 哺乳類の血液から血液由来成長因子含有組成物を調製する方法であって、金属イオンとキレート錯体を形成し得る抗凝固剤を前記血液に添加する工程と、前記抗凝固剤が添加された前記血液からバフィーコート成分を含む多血小板血漿を分離する工程と、前記多血小板血漿を凍結融解して活性化させる工程と、を含み、前記多血小板血漿を分離する工程が、前記抗凝固剤が添加された前記血液について5~2000G、1~60分による第1の遠心分離処理を施して血漿及びバフィーコートを含む上層部を回収するステップを含み、且つ、前記上層部を回収するステップにおいて、全液量の体積を基準として、回収する前記上層部の体積比が、1:0.1~0.5である方法;
〔2〕 前記上層部を回収するステップの後に、さらに、前記上層部について100~2500G、1~30分による第2の遠心分離処理を施して血小板及びバフィーコート成分をペレット化するステップ;及び上清を除去し、前記血小板及び前記バフィーコート成分を懸濁して多血小板血漿を得るステップ;を含み、且つ、
前記多血小板血漿を得るステップにおいて、ペレットの体積を基準として、体積比が1:1~10の体積量の上清を残しそれ以外を除去する、前記〔1〕に記載の方法;
〔3〕 前記第1の遠心分離処理が、100~1000G、1~30分であり、且つ、前記第2の遠心分離処理が、1000~2500G、1~30分である、前記〔2〕に記載の方法;
〔4〕 前記抗凝固剤が、クエン酸、EDTA、又はこれらの塩を含む、前記〔1〕~〔3〕いずれかに記載の方法;
〔5〕 前記多血小板血漿を凍結融解して活性化させる工程が、-200℃~-20℃、10分以上の処理による凍結と、その後の20℃~50℃、10分以上の処理による融解により行われる、前記〔1〕~〔4〕いずれかに記載の方法;
〔6〕 前記多血小板血漿を分離する工程の前に、前記抗凝固剤を添加された血液を0~10℃で予冷蔵する工程をさらに含む、前記〔1〕~〔5〕いずれかに記載の方法;
〔7〕 孔径0.2~1μmのフィルターを用いて、活性化された前記多血小板血漿を濾過処理する工程をさらに含む、前記〔1〕~〔6〕いずれかに記載の方法;
〔8〕 活性化された前記多血小板血漿を凍結乾燥する工程をさらに含む、前記〔1〕~〔7〕いずれかに記載の方法;
〔9〕 哺乳類の血液から血液由来成長因子含有組成物を調製する方法であって、金属イオンとキレート錯体を形成し得る抗凝固剤を前記血液に添加する工程と、前記抗凝固剤が添加された前記血液からバフィーコート成分を含む多血小板血漿を分離する工程と、前記多血小板血漿を凍結融解して活性化させる工程と、孔径0.2~1μmのフィルターを用いて、活性化された前記多血小板血漿を濾過処理する工程と、を含む方法;
〔10〕 前記〔1〕~〔7〕及び〔9〕のいずれかに記載の方法により調製された血液由来成長因子含有組成物;
〔11〕 前記〔8〕に記載の方法により調製された血液由来成長因子含有凍結乾燥物;
が提供される。
本実施形態に係る血液由来成長因子含有組成物の調製方法は、哺乳類の血液から血液由来成長因子組成物を調製する方法であって、金属イオンとキレート錯体を形成し得る抗凝固剤を前記血液に添加する工程(以下、「添加工程」とも呼ぶ)と、前記抗凝固剤が添加された前記血液からバフィーコート成分を含む多血小板血漿を分離する工程(以下、「分離工程」とも呼ぶ)と、前記多血小板血漿を凍結融解して活性化させる工程(以下、「凍結融解工程」とも呼ぶ)と、を少なくとも含む方法である。ここで、「哺乳類の血液」とは、成長因子が含有されていれば特に制限されず、例えば、ヒト、ブタ、ウシ、ウマ、イヌ、ネコ及びサル等由来の血液であり、好ましくはヒト由来の血液である。また、前記「哺乳類の血液」は好ましくは自己由来である。以下、上述した各工程について詳述する。
本実施形態の方法においては、金属イオンとキレート錯体を形成する抗凝固剤が添加された血液を使用する。抗凝固剤は、採取された血液に後から添加してもよいが、予め抗凝固剤を含む採血管又は採血バッグ等の容器に血液を採取することが望ましい。
本実施形態の方法は、金属イオンとキレート錯体を形成する抗凝固剤が添加された血液から多血小板血漿を分離する工程の前に、抗凝固剤が添加された血液を予冷蔵する工程をさらに含んでもよい。血液を予冷蔵する工程における冷蔵温度は、例えば、0~10℃である。血液を予冷蔵する工程の工程時間は、例えば、0時間超72時間以下である。当該工程時間の下限値は、30分以上、1時間以上、2時間以上、4時間以上、8時間以上としてもよい。当該工程時間の上限値は、60時間以下、48時間以下、36時間以下、24時間以下としてもよい。
本実施形態の方法は、抗凝固剤が添加された血液からバフィーコート成分を含む多血小板血漿を分離する工程を含む。ここで、「バフィーコート」とは、血液を遠心分離したとき赤血球と血漿画分の中間にできる白血球を多く含んだ薄く白い層を指す。また、「バフィーコート成分を含む」とは、バフィーコートの少なくとも一部を含むことを意味する。
比較的低速度の第1の遠心分離処理は、5~2000G(通常の遠心分離機においては200~2000rpm程度に相当)の遠心分離であることが好ましい。回転条件は、100~1000G、150~750G、200~500Gとしてもよい。処理時間は、1~60分、1~30分、5~20分、10~15分としてもよい。
血小板及びバフィーコート成分が、第1の遠心分離処理後に回収した前記上層部について比較的高速度の第2の遠心分離処理によってペレット化される。
第2の遠心分離処理により得られたものから上清を除去し、その後、懸濁させることで、血小板及びバフィーコート成分を純化・濃縮することができる。ここで、上清を除去する工程では、任意の体積量を除去できる。具体的には、例えば、ペレットの体積を基準として、体積比が好ましくは1:1~10であり、より好ましくは1:1~5であり、さらに好ましくは1:1~3の体積量の上清を残しそれ以外を除去できる。
本工程によって、多血小板血漿が活性化され、多数の成長因子及び抗炎症性サイトカインが放出される。
本実施形態の方法は、多血小板血漿から血液細胞を除去する工程をさらに含んでもよい。
本実施形態の方法は、多血小板血漿を凍結乾燥する工程をさらに含んでもよい。
本実施形態に係る血液由来成長因子含有組成物は、哺乳類の血液から調製された血液由来成長因子含有組成物であって、特定の成長因子の含有量が極めて高い血液由来成長因子含有組成物である。当該血液由来成長因子含有組成物は液体、半固体、又は乾燥固体であってもよい。以下、含有成分について詳述する。
(VEGF)
本実施形態による血液由来成長因子含有組成物は、成長因子VEGFの含有量が好ましくは90pg/mL以上であり、より好ましくは100pg/mL以上であり、さらに好ましくは200pg/mL以上であり、特に好ましくは300pg/mL以上である。VEGFの含有量の上限値は、血液を採取した哺乳類の個体差によって変わるが、例えば10000pg/mLである。ここで、本明細書及び本特許請求の範囲における「pg/mL」は、濾過処理工程後の多血小板血漿における濃度であり、又は凍結乾燥工程後の多血小板血漿を、溶媒1mLあたり300mg融解した多血小板血漿における濃度である。VEGFは、血小板にも含まれるが、バフィーコートに多く含まれる成長因子である。VEGFは血管新生作用があり、VEGFを多く含む血液由来成長因子含有組成物は、より高い創傷治癒効果が期待できる。また、当該組成物の乾燥質量に対する、VEGFの含有量(pg/乾燥物1g)は、好ましくは300pg/g以上であり、より好ましくは700pg/g以上であり、さらに好ましくは1000pg/g以上である。当該組成物の乾燥質量に対する、VEGFの含有量の上限値は、血液を採取した哺乳類の個体差によって変わるが、例えば30000pg/gである。ここで、当該組成物が乾燥形態でない場合には、当該組成物の乾燥質量は、下記乾燥条件にて当該組成物を乾燥させた後のものの質量である。
(乾燥条件)
FDU-1110(東京理化器械株式会社製)を用いて、5Pa、-45℃の条件下、16時間に亘って、乾燥対象の組成物に対して真空凍結乾燥処理を施す。
本実施形態による血液由来成長因子含有組成物は、他の成長因子を含んでもよい。成長因子の種類は、特に限定されないが、例えば、血小板由来成長因子(例えばPDGF-AB及びPDGF-BB)、上皮成長因子(EGF)、線維芽細胞増殖因子(FGF)、トランスフォーミング増殖因子(例えばTGF-β)、インスリン様成長因子(IGF)、肝細胞成長因子(HGF)である。
(1L-1Ra)
本実施形態による血液由来成長因子含有組成物は、抗炎症性サイトカイン:インターロイキン-1レセプターアンタゴニスト(Interleukin-1 receptor antagonist:以下、「IL-1Ra」とも呼ぶ)を含んでもよい。IL-1Raの含有量は、好ましくは100pg/mL以上であり、より好ましくは150pg/mL以上であり、さらに好ましくは200pg/mL以上である。IL-1Raの含有量の上限値は、血液を採取した哺乳類の個体差によって変わるが、例えば50000pg/mLである。IL-1Raは主に白血球に含まれる。IL-1Raは、炎症性サイトカインIL-1α及びIL-1βのアンタゴニストとして作用し、炎症を抑制する。また、当該組成物の乾燥質量に対する、抗炎症性サイトカインIL-1Raの含有量(pg/乾燥物1g)は、好ましくは300pg/g以上であり、より好ましくは450pg/g以上であり、さらに好ましくは600pg/g以上である。当該組成物の乾燥質量に対する、IL-1Raの含有量の上限値は、血液を採取した哺乳類の個体差によって変わるが、例えば150000pg/gである。ここで、当該組成物が乾燥形態でない場合には、当該組成物の乾燥質量は上記乾燥条件にて当該組成物を乾燥させた後のものの質量である。
本実施形態による血液由来成長因子含有組成物は、炎症性サイトカイン:インターロイキン-1β(Interleukin-1β:以下、「IL-1β」とも呼ぶ)、インターロイキン-6(Interleukin-6:以下、「IL-6」とも呼ぶ)、及び腫瘍壊死因子(Tumor necrosis factor-α:以下、「TNF-α」とも呼ぶ)を含み得る。IL-1β及びIL-6の含有量はそれぞれ独立に、好ましくは10pg/mL以下であり、より好ましくは9.5pg/mL以下であり、さらに好ましくは9.0pg/mL以下である。TNF-αの含有量は、好ましくは20pg/mL以下であり、より好ましくは19pg/mL以下であり、さらに好ましくは18pg/mL以下である。これらの炎症性サイトカインの過剰な発現は、関節リウマチなどの疾患発症を招くおそれがある。本実施形態による血液由来成長因子含有組成物に含まれる炎症性サイトカインは上記範囲のような低濃度にすることで、人体や動物に投与してもこのような副作用を引き起こしにくい。
また、当該組成物の乾燥質量に対する、IL-1β及びIL-6及びの含有量(pg/乾燥物1g)はそれぞれ独立に、好ましくは30pg/g以下であり、より好ましくは28pg/g以下であり、さらに好ましくは27pg/g以下である。さらに、当該組成物の乾燥質量に対する、TNF-αの含有量(pg/乾燥物1g)は、好ましくは60pg/g以下であり、より好ましくは57pg/g以下であり、さらに好ましくは54pg/g以下である。ここで、当該組成物が乾燥形態でない場合には、当該組成物の乾燥質量は上記乾燥条件にて当該組成物を乾燥させた後のものの質量である。
(成長因子と抗炎症性サイトカイン)
本実施形態による血液由来成長因子含有組成物は、好適には、成長因子と抗炎症性サイトカインをバランスよく含む。具体的には、成長因子VEGFの質量を基準とした、抗炎症性サイトカインIL-1Raの質量比が、好ましくは1:1~50であり、より好ましくは1:1~15であり、さらに好ましくは1:1~10である。このような比率で成長因子と抗炎症性サイトカインを含む血液由来成長因子含有組成物は、創傷治癒効果と炎症抑制効果を併せ持つため、人体や動物に投与したときにより高い治療効果を奏する。
本実施形態による血液由来成長因子含有組成物は、好適には、成長因子を多く含みつつ、炎症性サイトカインをほとんど含まない。具体的には、成長因子VEGFの質量を基準とした、炎症性サイトカインIL-1βの質量比が、好ましくは1:0.001~0.05であり、より好ましくは1:0.001~0.03であり、さらに好ましくは1:0.001~0.01である。このような比率で成長因子と炎症性サイトカインを含む血液由来成長因子含有組成物は、人体や動物に投与したときに炎症を引き起こしにくく、高い治療効果を奏する。
本実施形態による血液由来成長因子含有組成物は、凍結乾燥状態にあってもよい。
(VEGF)
本実施形態による凍結乾燥状態にある血液由来成長因子含有組成物は、成長因子VEGFの含有量が好ましくは300pg/g以上であり、より好ましくは700pg/g以上であり、さらに好ましくは1000pg/g以上である。VEGFの含有量の上限値は、血液を採取した哺乳類の個体差によって変わるが、例えば30000pg/gである。
本実施形態による凍結乾燥状態にある血液由来成長因子含有組成物は、他の成長因子を含んでもよい。成長因子の種類は、特に限定されないが、例えば、血小板由来成長因子(例えばPDGF-AB及びPDGF-BB)、上皮成長因子(EGF)、線維芽細胞増殖因子(FGF)、トランスフォーミング増殖因子(例えばTGF-β)、インスリン様成長因子(IGF)、肝細胞成長因子(HGF)である。
(1L-1Ra)
本実施形態による凍結乾燥状態にある血液由来成長因子含有組成物は、抗炎症性サイトカインIL-1Raを含んでもよい。IL-1Raの含有量は、好ましくは300pg/g以上であり、より好ましくは450pg/g以上であり、さらに好ましくは600pg/g以上である。IL-1Raの含有量の上限値は、血液を採取した哺乳類の個体差によって変わるが、例えば150000pg/gである。
本実施形態による血液由来成長因子含有組成物は、炎症性サイトカインIL-1β、IL-6及びTNF-αを含み得る。IL-1β及びIL-6の含有量はそれぞれ独立に、好ましくは30pg/g以下であり、より好ましくは28pg/g以下であり、さらに好ましくは27pg/g以下である。また、TNF-αの含有量(pg/乾燥物1g)は、好ましくは60pg/g以下であり、より好ましくは57pg/g以下であり、さらに好ましくは54pg/g以下である。
本実施形態による凍結乾燥状態にある血液由来因子含有組成物は、好適には、成長因子と抗炎症性サイトカインをバランスよく含む。具体的には、成長因子VEGFの質量を基準とした、抗炎症性サイトカインIL-1Raの質量比が、好ましくは1:1~50であり、より好ましくは1:1~15であり、さらに好ましくは1:1~10である。また、当該血液由来成長因子含有組成物は、好適には、成長因子を多く含みつつ、炎症性サイトカインをほとんど含まない。具体的には、成長因子VEGFの質量を基準とした、炎症性サイトカインIL-1βの質量比が、好ましくは1:0.001~0.05であり、より好ましくは1:0.001~0.03であり、さらに好ましくは1:0.001~0.01である。
(実施例1~5の作製)
男性5人からそれぞれ約34mLの全血をACD溶液入り採血管(BDバイキュアナ採血管 日本ベクトン・ディッキンソン株式会社#364606)に適宜採取した。これらを恒温槽において4℃で一晩(約16時間)冷蔵保存した。保存後、第1の遠心分離処理{1200rpm(280G相当)、10分間、24℃}を行い、血漿画分全て及びバフィーコートを含む上層部(15mL:全液量の体積を基準として、回収した体積比は1:0.4)を回収した。この血漿について第2の遠心分離処理{2680rpm(1400G相当)、10分間、24℃}を行い、ペレットと上清に分離した。分離後、ペレットの体積を基準として、体積比が1:2となる上清は残し、それ以外の上清(血漿)を除去した。残った上清によってペレットを懸濁させ、懸濁液(多血小板血漿)を得た。
実施例1~5の試料について、乾燥物300mgに対して注射用水を1mL加え融解し、ELISA法によって成長因子、抗炎症性サイトカイン、及び炎症性サイトカインの量/濃度を解析した。その結果を表1に示す。VEGF、IL-1Ra、IL-1β、IL-6及びTNF-αの含有量はそれぞれ100pg/mL以上、100pg/mL以上、10pg/mL以下、10pg/mL以下及び20pg/mL以下であった。
また、これより算出された成長因子、抗炎症性サイトカイン、及び炎症性サイトカインの量/乾燥物(1g)の結果を表2に示す。
(実施例6~10の作製)
男性1人から約85mLの全血をACD溶液入り採血管に採取し、5本に分注した。こうして検体6~10を得た。その後、検体7及び8は、冷蔵庫において4℃で、それぞれ約24時間及び約48時間に亘り冷蔵した。また、検体9及び10は、室温(25℃)で、それぞれ約24時間及び約48時間に亘り静置した。これら検体6~10について、実施例1~5と同様に、多血小板血漿を回収する工程、凍結融解し活性化する工程及び、凍結乾燥する工程を施し、実施例6~10の試料を得た。
実施例6~10の試料について、乾燥物300mgに対して注射用水を1mL加え融解し、ELISA法によって成長因子(VEGF)、抗炎症性サイトカイン(IL-1Ra)の量/濃度を解析した。採血後、予冷蔵を経ずに作製したもの(実施例6)を100%としたときの各サイトカイン量比を図1に示す。4℃で24時間(実施例7)又は48時間(実施例8)予冷蔵した場合、各因子の含有量がIL-1Raは約500%、VEGFは約200%に増加していた。一方、予冷蔵に代えて、室温に24時間(実施例9)又は48時間(実施例10)静置した場合は、各因子の顕著な増加は確認されなかった。
(サンプルの作製)
男性1人(検体11)及び女性1人(検体12)からそれぞれ約76.5mLの全血をACD溶液入り採血管に採取し、9本に分注した。これらを恒温槽において4℃で一晩(約16時間)冷蔵保存した。保存後、表4に示す各分離条件に従って遠心分離し、懸濁液(多血小板血漿)を得た。このとき、第1の遠心分離後の回収した上層部の全液量に対する体積比及び(分離条件Bを除く)第2の遠心分離後の除去した上清量は、実施例1~5と同様の条件とした。また、このとき、比較のために、分離条件Bを除く各分離条件について、第1の遠心分離後に血漿画分のみの上層部を回収した、バフィーコート成分を含まない懸濁液も取得した。取得した各サンプルについて、実施例1~5と同様に、凍結融解し活性化する工程、血液細胞を除去する工程及び凍結乾燥する工程を施した。
各サンプルについて、乾燥物300mgに対して注射用水を1mL加え融解し、ELISA法によって成長因子(VEGF)、抗炎症性サイトカイン(IL-1Ra)の量/濃度を解析した。その結果を、図2(VEGF)及び図3(IL-1Ra)に示す。図2及び図3より、バフィーコート成分を含むサンプルは、分離条件によらず、VEGF及びIL-1Raを多く含む血液由来成長因子含有組成物を製造できることが分かった。
Claims (11)
- 哺乳類の血液から血液由来成長因子含有組成物を調製する方法であって、
金属イオンとキレート錯体を形成し得る抗凝固剤を前記血液に添加する工程と、
前記抗凝固剤が添加された前記血液からバフィーコート成分を含む多血小板血漿を分離する工程と、
前記多血小板血漿を凍結融解して活性化させる工程と、
を含み、
前記多血小板血漿を分離する工程が、
前記抗凝固剤が添加された前記血液について5~2000G、1~60分による第1の遠心分離処理を施して血漿及びバフィーコートを含む上層部を回収するステップを含み、且つ、
前記上層部を回収するステップにおいて、全液量の体積を基準として、回収する前記上層部の体積比が、1:0.1~0.5である方法。 - 前記上層部を回収するステップの後に、さらに、前記上層部について100~2500G、1~30分による第2の遠心分離処理を施して血小板及びバフィーコート成分をペレット化するステップ;及び上清を除去し、前記血小板及び前記バフィーコート成分を懸濁して多血小板血漿を得るステップ;を含み、且つ、
前記多血小板血漿を得るステップにおいて、ペレットの体積を基準として、体積比が1:1~10の体積量の上清を残しそれ以外を除去する、請求項1記載の方法。 - 前記第1の遠心分離処理が、100~1000G、1~30分であり、且つ、前記第2の遠心分離処理が、1000~2500G、1~30分である、請求項2記載の方法。
- 前記抗凝固剤が、クエン酸、EDTA、又はこれらの塩を含む、請求項1~3のいずれか1項記載の方法。
- 前記多血小板血漿を凍結融解して活性化させる工程が、-200℃~-20℃、10分以上の処理による凍結と、その後の20℃~50℃、10分以上の処理による融解により行われる、請求項1~4のいずれか1項記載の方法。
- 前記多血小板血漿を分離する工程の前に、前記抗凝固剤を添加された血液を0~10℃で予冷蔵する工程をさらに含む、請求項1~5のいずれか1項記載の方法。
- 孔径0.2~1μmのフィルターを用いて、活性化された前記多血小板血漿を濾過処理する工程をさらに含む、請求項1~6のいずれか1項記載の方法。
- 活性化された前記多血小板血漿を凍結乾燥する工程をさらに含む、請求項1~7のいずれか1項記載の方法。
- 哺乳類の血液から血液由来成長因子含有組成物を調製する方法であって、
金属イオンとキレート錯体を形成し得る抗凝固剤を前記血液に添加する工程と、
前記抗凝固剤が添加された前記血液からバフィーコート成分を含む多血小板血漿を分離する工程と、
前記多血小板血漿を凍結融解して活性化させる工程と、
孔径0.2~1μmのフィルターを用いて、活性化された前記多血小板血漿を濾過処理する工程と、
を含む方法。 - 請求項1~7及び9のいずれか1項記載の方法により調製された血液由来成長因子含有組成物。
- 請求項8に記載の方法により調製された血液由来成長因子含有凍結乾燥物。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020237042292A KR20240007208A (ko) | 2021-05-14 | 2022-05-13 | 혈액 유래 성장 인자 함유 조성물 및 그 조제 방법 |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2021082782A JP7025070B1 (ja) | 2021-05-14 | 2021-05-14 | 血液由来成長因子含有組成物及びその調製方法 |
JP2021-082782 | 2021-05-14 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2022239870A1 true WO2022239870A1 (ja) | 2022-11-17 |
Family
ID=81124371
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2022/020281 WO2022239870A1 (ja) | 2021-05-14 | 2022-05-13 | 血液由来成長因子含有組成物及びその調製方法 |
Country Status (4)
Country | Link |
---|---|
JP (1) | JP7025070B1 (ja) |
KR (1) | KR20240007208A (ja) |
TW (1) | TW202310856A (ja) |
WO (1) | WO2022239870A1 (ja) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2024122618A1 (ja) * | 2022-12-07 | 2024-06-13 | セルソース株式会社 | 成長因子含有組成物の調製方法 |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP7175055B1 (ja) | 2021-05-14 | 2022-11-18 | セルソース株式会社 | 血液由来成長因子含有組成物及びその調製方法 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2012505239A (ja) * | 2008-10-09 | 2012-03-01 | バイオパラドックス,リミテッド ライアビリティー カンパニー | 心臓治療用の多血小板血漿製剤 |
JP2013209409A (ja) * | 2007-03-07 | 2013-10-10 | Jms Co Ltd | 血清調製装置 |
JP2016164155A (ja) * | 2015-02-26 | 2016-09-08 | 株式会社細胞応用技術研究所 | 多血小板血漿の保存方法 |
JP2017171597A (ja) * | 2016-03-22 | 2017-09-28 | 株式会社細胞応用技術研究所 | 血小板濃縮製剤の製造方法 |
JP2017533896A (ja) * | 2014-09-30 | 2017-11-16 | アントノール リミテッド | 自己由来の生理学的流体から、強化された抗炎症性/抗異化性物質および再生性物質を作製するための方法および組成物 |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7473678B2 (en) | 2004-10-14 | 2009-01-06 | Biomimetic Therapeutics, Inc. | Platelet-derived growth factor compositions and methods of use thereof |
WO2012030593A2 (en) * | 2010-09-03 | 2012-03-08 | Biomet Biologics, Llc | Methods and compositions for delivering interleukin-1 receptor antagonist |
TWI744300B (zh) * | 2016-03-23 | 2021-11-01 | 里爾中央醫學中心 | 改良性熱處理的血小板顆粒裂解液在製備用於治療神經系統疾病的組合物的用途 |
JP6840770B2 (ja) * | 2016-04-05 | 2021-03-10 | ゼネラル・エレクトリック・カンパニイ | 成長因子レベルを調節可能な活性化血小板組成物 |
EP3254684B1 (en) * | 2016-06-08 | 2019-10-23 | Lysatpharma GmbH | Human platelet lysate or fraction enriched in human platelet-derived extracellular vesicles, for use in medicine |
-
2021
- 2021-05-14 JP JP2021082782A patent/JP7025070B1/ja active Active
-
2022
- 2022-05-13 WO PCT/JP2022/020281 patent/WO2022239870A1/ja active Application Filing
- 2022-05-13 KR KR1020237042292A patent/KR20240007208A/ko unknown
- 2022-05-16 TW TW111118266A patent/TW202310856A/zh unknown
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2013209409A (ja) * | 2007-03-07 | 2013-10-10 | Jms Co Ltd | 血清調製装置 |
JP2012505239A (ja) * | 2008-10-09 | 2012-03-01 | バイオパラドックス,リミテッド ライアビリティー カンパニー | 心臓治療用の多血小板血漿製剤 |
JP2017533896A (ja) * | 2014-09-30 | 2017-11-16 | アントノール リミテッド | 自己由来の生理学的流体から、強化された抗炎症性/抗異化性物質および再生性物質を作製するための方法および組成物 |
JP2016164155A (ja) * | 2015-02-26 | 2016-09-08 | 株式会社細胞応用技術研究所 | 多血小板血漿の保存方法 |
JP2017171597A (ja) * | 2016-03-22 | 2017-09-28 | 株式会社細胞応用技術研究所 | 血小板濃縮製剤の製造方法 |
Non-Patent Citations (1)
Title |
---|
DE MELO BRUNA ALICE GOMES; MARTINS SHIMOJO ANDRéA ARRUDA; MARCELINO PEREZ AMANDA GOMES; DUARTE LANA JOSé FABIO SANTOS; A: "Distribution, recovery and concentration of platelets and leukocytes in L-PRP prepared by centrifugation", COLLOIDS AND SURFACES B: BIOINTERFACES, ELSEVIER AMSTERDAM, NL, vol. 161, 18 October 2017 (2017-10-18), NL , pages 288 - 295, XP085299369, ISSN: 0927-7765, DOI: 10.1016/j.colsurfb.2017.10.046 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2024122618A1 (ja) * | 2022-12-07 | 2024-06-13 | セルソース株式会社 | 成長因子含有組成物の調製方法 |
Also Published As
Publication number | Publication date |
---|---|
JP2022175958A (ja) | 2022-11-25 |
JP7025070B1 (ja) | 2022-02-24 |
KR20240007208A (ko) | 2024-01-16 |
TW202310856A (zh) | 2023-03-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2022239870A1 (ja) | 血液由来成長因子含有組成物及びその調製方法 | |
US5213814A (en) | Lyophilized and reconstituted blood platelet compositions | |
EP2884992B1 (en) | A method of preparing a growth factor concentrate derived from human platelets | |
JP6649257B2 (ja) | 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 | |
JPH10507770A (ja) | 血小板の貯蔵期間の延長法 | |
US20120183519A1 (en) | Treatment of erectile dysfunction using platelet-rich plasma | |
US20060039991A1 (en) | Biological tissue regenerative agent and method for preparing and using same | |
US4680177A (en) | Processes for the production of blood products | |
JP6391872B1 (ja) | 成長因子混合物およびその調製方法 | |
EP3068410B1 (en) | Method for obtaining a cytokine-rich composition and composition obtained by means of this method | |
JP7175055B1 (ja) | 血液由来成長因子含有組成物及びその調製方法 | |
JP6999927B2 (ja) | 多血小板血漿を製造する方法 | |
EP1848447B1 (en) | Biological tissue regenerative agent and method for preparing and using same | |
WO2024122618A1 (ja) | 成長因子含有組成物の調製方法 | |
WO1993007745A1 (en) | Method for freezing engrafting cells | |
JP3938973B2 (ja) | 細胞分離方法 | |
CN115125202B (zh) | 一种自体血细胞因子和外泌体血清及其制备方法 | |
MANCHANDA et al. | DIFFERENT TYPES OF PRP | |
US20220280606A1 (en) | Method of preparing a de-fibrinated platelet lysate, and uses of said method | |
GB2079292A (en) | Blood Clotting Factor Production | |
WO2021262803A1 (en) | Compositions and methods for treating hemorrhagic shock | |
JP2021109875A (ja) | 血小板溶解物の調製方法および雌性妊娠率を上げるための用途 | |
Cross | The Separation of Thromboplastin Formed from Pig’s Plasma in the Thromboplastin Generation Test |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 22807568 Country of ref document: EP Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 202317002462 Country of ref document: IN |
|
WWE | Wipo information: entry into national phase |
Ref document number: 12023553112 Country of ref document: PH |
|
ENP | Entry into the national phase |
Ref document number: 20237042292 Country of ref document: KR Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1020237042292 Country of ref document: KR |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 22807568 Country of ref document: EP Kind code of ref document: A1 |