CN115029308A - 干细胞外泌体制剂及其制备方法和应用 - Google Patents
干细胞外泌体制剂及其制备方法和应用 Download PDFInfo
- Publication number
- CN115029308A CN115029308A CN202210912539.9A CN202210912539A CN115029308A CN 115029308 A CN115029308 A CN 115029308A CN 202210912539 A CN202210912539 A CN 202210912539A CN 115029308 A CN115029308 A CN 115029308A
- Authority
- CN
- China
- Prior art keywords
- stem cell
- culture
- stem cells
- preparation
- mesenchymal stem
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 210000001808 exosome Anatomy 0.000 title claims abstract description 11
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims abstract description 34
- 102000004127 Cytokines Human genes 0.000 claims abstract description 18
- 108090000695 Cytokines Proteins 0.000 claims abstract description 18
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 claims abstract description 18
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 claims abstract description 17
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 claims abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 15
- BJDCWCLMFKKGEE-ISOSDAIHSA-N artenimol Chemical compound C([C@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2O[C@H](O)[C@@H]4C BJDCWCLMFKKGEE-ISOSDAIHSA-N 0.000 claims abstract description 14
- 229960002521 artenimol Drugs 0.000 claims abstract description 14
- 229930016266 dihydroartemisinin Natural products 0.000 claims abstract description 14
- 102100021943 C-C motif chemokine 2 Human genes 0.000 claims abstract description 13
- 101710155857 C-C motif chemokine 2 Proteins 0.000 claims abstract description 13
- 238000012258 culturing Methods 0.000 claims abstract description 13
- 239000001963 growth medium Substances 0.000 claims abstract description 13
- 206010021143 Hypoxia Diseases 0.000 claims abstract description 12
- 102100039364 Metalloproteinase inhibitor 1 Human genes 0.000 claims abstract description 12
- 108010031374 Tissue Inhibitor of Metalloproteinase-1 Proteins 0.000 claims abstract description 12
- 108090001005 Interleukin-6 Proteins 0.000 claims abstract description 8
- 108090001007 Interleukin-8 Proteins 0.000 claims abstract description 8
- 239000012228 culture supernatant Substances 0.000 claims abstract description 7
- -1 TGF-1 Proteins 0.000 claims abstract description 6
- 238000004113 cell culture Methods 0.000 claims abstract description 6
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 claims abstract 3
- 229910052760 oxygen Inorganic materials 0.000 claims description 23
- 239000001301 oxygen Substances 0.000 claims description 17
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 16
- 230000029663 wound healing Effects 0.000 claims description 14
- 210000003954 umbilical cord Anatomy 0.000 claims description 11
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 10
- 239000002609 medium Substances 0.000 claims description 8
- 239000007640 basal medium Substances 0.000 claims description 5
- 239000003814 drug Substances 0.000 claims description 5
- 230000008439 repair process Effects 0.000 claims description 5
- 230000001737 promoting effect Effects 0.000 claims description 4
- 229940079593 drug Drugs 0.000 claims description 3
- 230000007954 hypoxia Effects 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims 2
- 238000012136 culture method Methods 0.000 abstract description 14
- 230000001146 hypoxic effect Effects 0.000 abstract description 9
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 description 15
- 206010052428 Wound Diseases 0.000 description 13
- 208000027418 Wounds and injury Diseases 0.000 description 13
- 210000004027 cell Anatomy 0.000 description 10
- 230000028327 secretion Effects 0.000 description 9
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 8
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 7
- 102000004889 Interleukin-6 Human genes 0.000 description 6
- 102000004890 Interleukin-8 Human genes 0.000 description 6
- 102000015696 Interleukins Human genes 0.000 description 6
- 108010063738 Interleukins Proteins 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 5
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 229960005070 ascorbic acid Drugs 0.000 description 4
- 235000010323 ascorbic acid Nutrition 0.000 description 4
- 239000011668 ascorbic acid Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000013508 migration Methods 0.000 description 4
- 210000003491 skin Anatomy 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 229930182816 L-glutamine Natural products 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- 230000035876 healing Effects 0.000 description 3
- 230000005012 migration Effects 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 238000010998 test method Methods 0.000 description 3
- 230000017423 tissue regeneration Effects 0.000 description 3
- 235000013343 vitamin Nutrition 0.000 description 3
- 229940088594 vitamin Drugs 0.000 description 3
- 229930003231 vitamin Natural products 0.000 description 3
- 239000011782 vitamin Substances 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 238000008157 ELISA kit Methods 0.000 description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 230000033115 angiogenesis Effects 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 210000000981 epithelium Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 238000005469 granulation Methods 0.000 description 2
- 230000003179 granulation Effects 0.000 description 2
- 210000002510 keratinocyte Anatomy 0.000 description 2
- 239000012982 microporous membrane Substances 0.000 description 2
- 239000007758 minimum essential medium Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 230000002980 postoperative effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 231100000241 scar Toxicity 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 230000009772 tissue formation Effects 0.000 description 2
- QDGAVODICPCDMU-UHFFFAOYSA-N 2-amino-3-[3-[bis(2-chloroethyl)amino]phenyl]propanoic acid Chemical compound OC(=O)C(N)CC1=CC=CC(N(CCCl)CCCl)=C1 QDGAVODICPCDMU-UHFFFAOYSA-N 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 102000007644 Colony-Stimulating Factors Human genes 0.000 description 1
- 108010071942 Colony-Stimulating Factors Proteins 0.000 description 1
- FFEARJCKVFRZRR-UHFFFAOYSA-N L-Methionine Natural products CSCCC(N)C(O)=O FFEARJCKVFRZRR-UHFFFAOYSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- 229930182844 L-isoleucine Natural products 0.000 description 1
- 235000019454 L-leucine Nutrition 0.000 description 1
- 239000004395 L-leucine Substances 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 229930195722 L-methionine Natural products 0.000 description 1
- 102000005741 Metalloproteases Human genes 0.000 description 1
- 108010006035 Metalloproteases Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000006909 anti-apoptosis Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 description 1
- 229960002327 chloral hydrate Drugs 0.000 description 1
- 230000037319 collagen production Effects 0.000 description 1
- 229940047120 colony stimulating factors Drugs 0.000 description 1
- 210000005069 ears Anatomy 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000037313 granulation tissue formation Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 229960002885 histidine Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 229940047122 interleukins Drugs 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 229960003136 leucine Drugs 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 229940078752 magnesium ascorbyl phosphate Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229960004452 methionine Drugs 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- IFGCUJZIWBUILZ-UHFFFAOYSA-N sodium 2-[[2-[[hydroxy-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyphosphoryl]amino]-4-methylpentanoyl]amino]-3-(1H-indol-3-yl)propanoic acid Chemical compound [Na+].C=1NC2=CC=CC=C2C=1CC(C(O)=O)NC(=O)C(CC(C)C)NP(O)(=O)OC1OC(C)C(O)C(O)C1O IFGCUJZIWBUILZ-UHFFFAOYSA-N 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- WPLOVIFNBMNBPD-ATHMIXSHSA-N subtilin Chemical compound CC1SCC(NC2=O)C(=O)NC(CC(N)=O)C(=O)NC(C(=O)NC(CCCCN)C(=O)NC(C(C)CC)C(=O)NC(=C)C(=O)NC(CCCCN)C(O)=O)CSC(C)C2NC(=O)C(CC(C)C)NC(=O)C1NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C1NC(=O)C(=C/C)/NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)CNC(=O)C(NC(=O)C(NC(=O)C2NC(=O)CNC(=O)C3CCCN3C(=O)C(NC(=O)C3NC(=O)C(CC(C)C)NC(=O)C(=C)NC(=O)C(CCC(O)=O)NC(=O)C(NC(=O)C(CCCCN)NC(=O)C(N)CC=4C5=CC=CC=C5NC=4)CSC3)C(C)SC2)C(C)C)C(C)SC1)CC1=CC=CC=C1 WPLOVIFNBMNBPD-ATHMIXSHSA-N 0.000 description 1
- 229960002898 threonine Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 210000003014 totipotent stem cell Anatomy 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- HTJNEBVCZXHBNJ-XCTPRCOBSA-H trimagnesium;(2r)-2-[(1s)-1,2-dihydroxyethyl]-3,4-dihydroxy-2h-furan-5-one;diphosphate Chemical compound [Mg+2].[Mg+2].[Mg+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.OC[C@H](O)[C@H]1OC(=O)C(O)=C1O HTJNEBVCZXHBNJ-XCTPRCOBSA-H 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 210000002444 unipotent stem cell Anatomy 0.000 description 1
- 229960004295 valine Drugs 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord 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
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/02—Atmosphere, e.g. low oxygen conditions
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/44—Thiols, e.g. mercaptoethanol
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Developmental Biology & Embryology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Cell Biology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Dermatology (AREA)
- Rheumatology (AREA)
- Biochemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Virology (AREA)
- Epidemiology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明属于干细胞培养技术领域,具体涉及富含细胞因子的干细胞外泌体制剂的制备方法和应用,该方法包括:在低氧的条件下培养干细胞;和或在含双氢青蒿素的培养基中培养干细胞;收集源自上述干细胞培养液的培养上清。采用上述培养方法培养的间充质干细胞高度表达IL‑6、IL‑8、TGF‑1、MCP‑1、VEGF、TIMP‑1和GM‑CSF因子,特别是其他培养方法中低表达或者是不表达的VEGF和GM‑CSF因子含量显著增加。
Description
技术领域
本发明属于干细胞培养技术领域。更具体地,涉及干细胞外泌体制剂及其制备方法和应用。
背景技术
已知多种细胞因子参与了组织损失修复的过程,其中,包括趋化因子,白细胞介素,生长因子和集落刺激因子等,在上述细胞因子中,发挥关键作用的主要有白细胞介素因子、TGF-1、MCP-1、VEGF、TIMP-1和GM-CSF等。其中,白细胞介素因子能够促进角质细胞增殖和迁移、促进上皮细胞组织再生和瘢痕组织形成;TGF-1可促进中性粒细胞、巨噬细胞迁移,刺激成纤维细胞增殖和瘢痕组织形成;MCP-1能够促进伤口部位上皮组织再生、血管发生和胶原蛋白的生成;VEGF能够促进伤口处血管生成和肉芽组织的形成;GM-CSF能够促进角质细胞分化、刺激内皮细胞增殖和迁移、加速伤口上皮细胞组织的再生;TIMP-1可以中和金属蛋白酶的作用,促进伤口的愈合。增强或提高间充质干细胞分泌上述细胞因子的能力可以改善伤口愈合状况,具有非常重要的临床意义。
然而,已有研究表明,现有培养的脐带间充质干细胞培养上清液中多表达白细胞介素因子、MCP-1和TIMP-1,但是对于GM-CSF和VEGF表达较低或者不表达。
发明内容
本发明涉及培养间充质干细胞的方法,该间充质干细胞能够同时高度表达TGF-1、GM-CSF和VEGF等伤口愈合相关因子;更一进步地,本发明涉及富含细胞因子的外泌体制剂,该外泌体制剂收集源自上述间充质干细胞培养液的培养上清;更进一步地,本发明涉及该外泌体制备促进皮肤修复、创伤愈合药物的用途。
具体而言,本发明的一个方面提供了培养高度表达细胞因子的间充质干细胞群的方法,第一种培养方法,包括:在低氧的条件下培养干细胞。已知低氧条件不会影响干细胞的表型及干性,并可促进干细胞增殖、分化、迁徙、抗凋亡及对缺血缺氧的耐受,但在申请日前,仍然不知低氧条件对间充质干细胞分泌伤口愈合相关因子效应的影响如何。根据本发明,低氧条件可通过低氧装置控制实现,在低氧条件下,氧气的浓度可以为1~3%,最好是1%、2%或3%。
在本发明具有代表性的实施例中,用于在低氧条件下培养间充质干细胞的培养基中可以包括加入或者未加入胎牛血清的基础培养基;基础培养基的例子包括DMEM、MEM或DMEM/F12;根据需要还可含有氨基酸、维生素、抗生素等活性成分。
氨基酸的例子包括:L-谷氨酰胺、L-组氨酸、L-异亮氨酸、L-亮氨酸、L-赖氨酸、L-蛋氨酸、L-苯丙氨酸、L-苏氨酸、L-色氨酸、L-缬氨酸等。在本发明的实施例中最好使用L-谷氨酰胺,L-谷氨酰胺的浓度通常介乎1~3mmol/L之间,例如2mmol/L。
维生素的例子包括:抗坏血酸或其衍生物,如抗坏血酸磷酸酯镁;在本发明的实施例中最好使用抗坏血酸,在培养基中,抗坏血酸的浓度通常介乎1~5mg/ml之间,例如约2mg/ml。
抗生素的例子包括链霉素和/或青霉素,当含有抗生素时,可以不包含其他添加剂成分,在本发明的实施例中最好使用链霉素和青霉素,青霉素的浓度通常介乎50~120U/ml之间,例如100U/ml。链霉素的浓度通常介乎于50~120μg/ml,例如100μg/ml。
本发明表1显示了2%O2、3%O2和常氧条件下培养的人脐带间充质干细胞细胞因子分泌情况,从结果可看出,低氧条件培养相关因子的表达量均比常氧条件下要高,特别是IL-6、IL-8和MCP-1表达量与常氧相比显著增加(P<0.05);但是GM-CSF和VEGF因子无论是在低氧或者常氧条件下的表达量均较低。
本发明另一方面提供了培养高度表达细胞因子的间充质干细胞群的第二种方法,包括:在含双氢青蒿素的培养基中培养干细胞;在申请日之前,尚未报道双氢青蒿素用于干细胞的培养,并刺激其高度表达相关细胞因子的例子。
特别的,在培养基中,双氢青蒿素的浓度通常介乎1~10μmol/L之间,例如3μmol/L、5μmol/L或6μmol/L。
特别的,在本发明具有代表性的实施例中,含有双氢青蒿素的培养基还可以进一步包含适量的基础培养基和2-巯基乙醇。基础培养基的例子包括DMEM、MEM或DMEM/F12,在本发明的实施例中最好使用DMEM基础培养基。
在本发明具有代表性的实施例中,2-巯基乙醇的存在是必要的,在培养基中,2-巯基乙醇的浓度通常介乎1~5μl之间,例如2μl或3μl。
特别的,可以不包含胎牛血清或者其他添加剂组分,如氨基酸、维生素、抗生素等。
本文中所述“干细胞”是指具有分化潜能和自我更新能力的细胞(全能干细胞、多能干细胞和单能干细胞),在本发明中特指间充质干细胞,更具体的是特指脐带间充质干细胞,在本文中已经验证了第一和第二培养方法均能够刺激人脐带间充质干细胞高度表达白细胞介素因子、MCP-1、TIMP-1、GM-CSF等相关伤口愈合因子,这种效应有可能在其他间充质干细胞,如骨髓间充质干细胞的身上同样可观察到,但还有待进一步研究验证。
特别的,本发明第一培养方法和第二培养方法可以单独执行或者组合或者同时执行。在本发明具有代表性的实施例中,第一培养方法和第二培养方法同时执行,将在获得的间充质干细胞身上观察极强的细胞因子分泌效应,特别是第一培养方法中低表达的GM-CSF和VEGF观察到强的分泌效应。
本发明表3显示了第一培养方法和第二培养方法同时执行培养的间充质干细胞相关因子的表达情况,通过比较加入或者未加入双氢青蒿素干预培养的间充质干细胞分泌细胞因子的情况,结果显示,2%O2下,加入双氢青蒿素干预培养的间充质干细胞同时高度表达IL-6、IL-8、TGF-1、MCP-1、VEGF、TIMP-1和GM-CSF,特别是常氧或低氧下低表达或者是不表达的VEGF和GM-CSF因子;经双氢青蒿素干预培养的间充质干细胞分泌的VEGF和GM-CS细胞因子量更高。
本发明另一方面提供一种外泌体制剂,该外泌体制剂收集源自第一培养方法和第二培养方法单独执行或者组合或者同时执行培养的间充质干细胞培养液的培养上清。已然通过试验证明,该培养上清中富含白细胞介素因子、MCP-1、TIMP-1、GM-CSF等相关伤口愈合因子,这些因子对于促进皮肤的修复、创面的愈合、上皮组织的形成是非常有利的。
本发明另一方面提供了富含细胞因子的外泌体制剂在制备促进皮肤修复、创伤愈合药物中的用途。这些药物可用于创伤、烧伤和溃疡愈合的临床治疗。
小鼠创面愈合试验显示,在不同的时间点,经注射第一培养方法和第二培养方法同时执行培养的间充质干细胞培养液上清的小鼠创面残余面积均显著低于其他组,创面愈合速率明显较其他组快,第7天可观察到新生肉芽,第14天时基本完成上皮化。
具体实施方式
以下结合具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
试验一、氧气浓度对人脐带间充质干细胞分泌相关细胞因子的影响研究
试验方法:
人脐带间充质干细胞的培养:取经鉴定符合间充质干细胞表型特征的P2代人脐带间充质干细胞,以1×104/cm2的密度接种于培养皿中,加入培养基(L-谷氨酰胺2mmol/L、抗坏血酸2mg/ml+余量DMEM),将培养皿放置于低氧装置中,以0.5L/min的流速通入混合气体(95%N2+5%CO2)来控制环境中的氧气浓度,分别于不同氧气浓度、37℃、体积分数5%CO2下培养96h,细胞达到80-90%融合后,收集上清液,离心去除死细胞和细胞碎片,微孔滤膜过滤,-20℃冻存,备用。
采用ELISA试剂盒按照说明书检测收集的上清液中IL-6、IL-8、TGF-1、MCP-1、VEGF、TIMP-1和GM-CSF含量,采用统计软件对数据进行统计分析,结果如下表1所示。
表1:
注:与常氧相比,*P<0.05;**P<0.01。
表1显示了2%O2、3%O2和常氧条件下培养的人脐带间充质干细胞细胞因子分泌情况,从结果可看出,低氧条件培养相关因子的表达量均比常氧条件下要高,特别是IL-6、IL-8、TIMP-1和MCP-1表达与常氧相比显著增加(P<0.05),但GM-CSF与VEGF的表达观察不到显著增加的趋势。
试验二、双氢青蒿素对人脐带间充质干细胞分泌相关细胞因子的影响研究
试验方法:取经鉴定符合间充质干细胞表型特征的P2代人脐带间充质干细胞,以1×104/cm2的密度接种于培养皿中,分别加入不同的培养基(表2),将培养皿放置于低氧装置中,以0.5L/min的流速通入混合气体(95%N2+5%CO2)来控制环境中的氧气浓度为2%,在37℃、体积分数5%CO2下培养96h,细胞达到80-90%融合后,收集上清液,离心去除死细胞和细胞碎片,微孔滤膜过滤,-20℃冻存,备用。
采用ELISA试剂盒按照说明书检测收集的上清液中IL-6、IL-8、TGF-1、MCP-1、VEGF、TIMP-1和GM-CSF含量,采用统计软件对数据进行统计分析,结果如下表3所示。
表2:
双氢青蒿素(μmol/L) | 2-巯基乙醇(μl) | DMEM培养基 | |
培养基A | 3μmol/L | 0 | 余量 |
培养基B | 0 | 3μl | 余量 |
培养基C | 3μmol/L | 3μl | 余量 |
培养基D | 5μmol/L | 2μl | 余量 |
表3:
与培养基C相比,*P<0.05;**P<0.01。
表3显示了2%O2下,加入或者未加入双氢青蒿素干预培养的间充质干细胞分泌细胞因子的情况,结果显示,加入双氢青蒿素干预培养的间充质干细胞同时高度表达IL-6、IL-8、TGF-1、MCP-1、VEGF、TIMP-1和GM-CSF,特别是常氧或低氧下低表达或者是不表达的VEGF和GM-CSF因子,经双氢青蒿素干预培养的间充质干细胞分泌量更高,与常氧条件相比,表达量增加了2倍左右。
试验三、对小鼠创面愈合的影响
试验方法:选用健康清洁级C57BL/6雄性小鼠50只,试验前12小时禁水禁食,用4%水合氯醛(0.01ml/g)腹腔注射麻醉小鼠,将其固定后除去小鼠背部毛发,将环形胶圈用尼龙线固定在小鼠背部中线两侧,距离小鼠耳朵2.5cm处,拉紧缝合线,使环形胶圈贴合在小鼠背部。使用皮肤打孔器在小鼠背部制作两个直径为1cm的创面,术后试验组分别经鼠尾静脉注射100μl试验例二制备的培养基A~D培养上清,空白组不做处理,分别于术后第0天、7天和14天观察创面愈合情况,拍照记录统计各时间点残余创面面积,创面愈合率(%)=(造模初期创面面积-术后不同时间点创面面积)/造模初期创面面积*100%,结果如下表4所示。
表4:
由表4可知,在不同的时间点,培养基C和培养基D组小鼠创面残余面积均显著低于其他组,创面愈合速率明显较另外三组快,第7天可观察到新生肉芽,第14天时基本完成上皮化。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
1.富含细胞因子的干细胞外泌体制剂的制备方法,其特征在于,该方法包括:
a1:在低氧的条件下培养干细胞;
a2:收集源自上述干细胞培养液的培养上清。
2.根据权利要求1所述制备方法,其特征在于,所述低氧条件中氧气的体积分数为1~3%。
3.根据权利要求1或2所述制备方法,其特征在于,该制备方法还包括:在含双氢青蒿素的培养基中培养干细胞。
4.根据权利要求3所述制备方法,其特征在于,所述培养基中,双氢青蒿素的浓度为1~10μmol/L。
5.根据权利要求3所述制备方法,其特征在于,所述培养基还包含1~5μl的2-巯基乙醇。
6.根据权利要求3所述制备方法,其特征在于,所述培养基还包含基础培养基。
7.根据权利要求1~6任一项所述制备方法,其特征在于,所述干细胞为脐带间充质干细胞。
8.根据权利要求1~6任一项所述制备方法,其特征在于,所述细胞因子包括IL-6、IL-8、TGF-1、MCP-1、VEGF、TIMP-1和GM-CSF。
9.根据权利要求1~8任一项所述制备方法制备得到的干细胞外泌体制剂。
10.根据权利要求9所述干细胞外泌体制剂在制备促进皮肤修复、创伤愈合药物中的用途。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210912539.9A CN115029308B (zh) | 2022-07-30 | 2022-07-30 | 干细胞外泌体制剂及其制备方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210912539.9A CN115029308B (zh) | 2022-07-30 | 2022-07-30 | 干细胞外泌体制剂及其制备方法和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN115029308A true CN115029308A (zh) | 2022-09-09 |
CN115029308B CN115029308B (zh) | 2023-06-09 |
Family
ID=83130806
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210912539.9A Active CN115029308B (zh) | 2022-07-30 | 2022-07-30 | 干细胞外泌体制剂及其制备方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN115029308B (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115491352A (zh) * | 2022-11-16 | 2022-12-20 | 广东先康达生物科技有限公司 | 促进干细胞外泌体分泌的培养液、外泌体制备及应用 |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101756957A (zh) * | 2008-12-26 | 2010-06-30 | 鼎泓国际投资(香港)有限公司 | 含有青蒿素及青蒿素类衍生物和组蛋白去乙酰化酶抑制剂的药物组合物及其应用 |
CN106701670A (zh) * | 2015-08-05 | 2017-05-24 | 朱轶 | 一种增强间充质干细胞分泌生物活性因子能力及培养液中活性因子的提取方法 |
CN106727500A (zh) * | 2017-01-13 | 2017-05-31 | 中国人民解放军白求恩国际和平医院 | 双氢青蒿素的应用、抑制药物及其应用 |
CN110876734A (zh) * | 2018-09-06 | 2020-03-13 | 杨昆德 | 包含胞外囊泡的制剂、用以制备该制剂的方法及其用途 |
WO2020070700A2 (en) * | 2018-10-04 | 2020-04-09 | Exogenus Therapeutics, Sa | Compositions comprising small extracellular vesicles derived from umbilical cord blood mononuclear cells with anti-inflammatory and immunomodulatory properties and process for obtaining them |
CN112076217A (zh) * | 2020-10-26 | 2020-12-15 | 重庆医科大学附属第一医院 | 一种间充质干细胞来源的抗炎症氧化脂质组合物的制备方法 |
CN114350603A (zh) * | 2022-01-23 | 2022-04-15 | 广州源康生物医药科技有限公司 | 包含外泌体的间充质干细胞胞外基质及其制备和在细胞修复中的应用 |
CN114699404A (zh) * | 2022-05-20 | 2022-07-05 | 北京大学口腔医学院 | 二氢青蒿素在制备促进骨组织再生修复药物中的应用 |
-
2022
- 2022-07-30 CN CN202210912539.9A patent/CN115029308B/zh active Active
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101756957A (zh) * | 2008-12-26 | 2010-06-30 | 鼎泓国际投资(香港)有限公司 | 含有青蒿素及青蒿素类衍生物和组蛋白去乙酰化酶抑制剂的药物组合物及其应用 |
CN106701670A (zh) * | 2015-08-05 | 2017-05-24 | 朱轶 | 一种增强间充质干细胞分泌生物活性因子能力及培养液中活性因子的提取方法 |
CN106727500A (zh) * | 2017-01-13 | 2017-05-31 | 中国人民解放军白求恩国际和平医院 | 双氢青蒿素的应用、抑制药物及其应用 |
CN110876734A (zh) * | 2018-09-06 | 2020-03-13 | 杨昆德 | 包含胞外囊泡的制剂、用以制备该制剂的方法及其用途 |
WO2020070700A2 (en) * | 2018-10-04 | 2020-04-09 | Exogenus Therapeutics, Sa | Compositions comprising small extracellular vesicles derived from umbilical cord blood mononuclear cells with anti-inflammatory and immunomodulatory properties and process for obtaining them |
CN112076217A (zh) * | 2020-10-26 | 2020-12-15 | 重庆医科大学附属第一医院 | 一种间充质干细胞来源的抗炎症氧化脂质组合物的制备方法 |
CN114350603A (zh) * | 2022-01-23 | 2022-04-15 | 广州源康生物医药科技有限公司 | 包含外泌体的间充质干细胞胞外基质及其制备和在细胞修复中的应用 |
CN114699404A (zh) * | 2022-05-20 | 2022-07-05 | 北京大学口腔医学院 | 二氢青蒿素在制备促进骨组织再生修复药物中的应用 |
Non-Patent Citations (3)
Title |
---|
LIPING TAN ET AL.: "Characteristics and regulation of mesenchymal stem cell plasticity by the microenvironment d specific factors involved in the regulation of MSC plasticity", 《GENES & DISEASES》 * |
叶锦豪等: "低氧预处理脐带间充质干细胞来源的外泌体对内皮细胞功能的影响", 《中国病理生理杂志》 * |
吴秀华: "二氢青高素抑制低氧环境多发性骨髓瘤RPMI8826细胞VEGF的表达和分泌及其诱导RPMI8226细胞凋亡", 《中国优秀博硕士学位论文全文数据库(硕士)医药卫生科技辑》 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115491352A (zh) * | 2022-11-16 | 2022-12-20 | 广东先康达生物科技有限公司 | 促进干细胞外泌体分泌的培养液、外泌体制备及应用 |
Also Published As
Publication number | Publication date |
---|---|
CN115029308B (zh) | 2023-06-09 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111773173B (zh) | 用于成脂分化诱导、脂肪组织再生、皮肤美白或改善皱纹的包含干细胞来源外泌体的组合物 | |
JP2013505011A (ja) | 幹細胞馴化培地組成物 | |
US20130058903A1 (en) | Stem-Cell Material and Method of Use | |
CN115029308B (zh) | 干细胞外泌体制剂及其制备方法和应用 | |
WO2020076739A1 (en) | Methods for the long-term expansion of granulocyte-macrophage progenitors and applications thereof | |
WO2021207025A1 (en) | Mesenchymal cell (msc) exosomes increase t cell differentiation towards t regulatory cells | |
CN111534477B (zh) | 小鼠肺组织原代上皮干细胞球培养方法 | |
CN112852713A (zh) | 一种人体皮肤成纤维细胞外泌体制备分离方法 | |
KR101656511B1 (ko) | 육모 및 탈모방지능이 개선된 지방줄기세포 배양액 및 그의 제조방법 | |
EP3226876A1 (en) | Stem cell material and method of manufacturing | |
CN113713176B (zh) | 一种水凝胶及其制备方法与应用 | |
KR102445484B1 (ko) | 장 오가노이드 제조용 배지 조성물 | |
US20230241121A1 (en) | Compositions and methods relating to exosomes derived from human dermal papilla cells | |
TWI640631B (zh) | Macrophage conditioned medium, dressing, preparation method and use thereof for promoting wound healing | |
KR20160064954A (ko) | 발모촉진 방법 | |
KR101673318B1 (ko) | 은나노 물질로 처리된 중간엽 줄기세포 또는 그 배양액을 유효성분으로 포함하는 상처 치료용 세포치료제 조성물 | |
Li et al. | Rat vibrissa dermal papilla cells promote healing of spinal cord injury following transplantation | |
WO2020179829A1 (ja) | アクチビンa及び/又はvpaを含む、nkt細胞培養のための添加剤、及びnkt細胞の培養方法 | |
US12006513B2 (en) | Methods for the long-term expansion of granulocyte-macrophage progenitors and applications thereof | |
Eby | The Role of Interleukin 4 Released from a 3D Matrix in Macrophage-Fibroblast Interactions Towards Inflammation and Fibrosis | |
CN112126622B (zh) | 一种可提高产量的脐带间充质干细胞原代分离培养方法 | |
KR20240019971A (ko) | 엑소좀 농도 향상을 위한 조성물 및 방법 | |
Li et al. | Biological characteristics of tissue engineered-nerve grafts enhancing peripheral nerve regeneration | |
CN104922698A (zh) | 人干细胞生长因子注射液及其制备方法 | |
CN117535237A (zh) | 人脐带间充质干细胞培养基及其制备方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
TA01 | Transfer of patent application right |
Effective date of registration: 20230519 Address after: Building 2, Beidaihe Life Science Park, Beidaihe New District, Qinhuangdao City, Hebei Province, 066100 Applicant after: Zhongbang Stem Cell Technology Co.,Ltd. Address before: Room C567, 2nd Floor, Unit 2, Building 2, No. 24, Jishan New Road Street, Tianhe District, Guangzhou City, Guangdong Province, 510000 Applicant before: Guangzhou Gaohua Biotechnology Co.,Ltd. |
|
TA01 | Transfer of patent application right | ||
GR01 | Patent grant | ||
GR01 | Patent grant |