CN111454898A - 一种间充质干细胞培养基用于化妆品的制备方法 - Google Patents
一种间充质干细胞培养基用于化妆品的制备方法 Download PDFInfo
- Publication number
- CN111454898A CN111454898A CN202010328316.9A CN202010328316A CN111454898A CN 111454898 A CN111454898 A CN 111454898A CN 202010328316 A CN202010328316 A CN 202010328316A CN 111454898 A CN111454898 A CN 111454898A
- Authority
- CN
- China
- Prior art keywords
- mesenchymal stem
- culture medium
- cells
- medium
- stem cell
- 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.)
- Pending
Links
- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 64
- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 239000006143 cell culture medium Substances 0.000 title claims abstract description 16
- 239000002537 cosmetic Substances 0.000 title claims abstract description 16
- 210000004027 cell Anatomy 0.000 claims abstract description 33
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 claims abstract description 28
- 239000003636 conditioned culture medium Substances 0.000 claims abstract description 24
- 239000000243 solution Substances 0.000 claims abstract description 24
- 239000001963 growth medium Substances 0.000 claims abstract description 20
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 8
- 239000012981 Hank's balanced salt solution Substances 0.000 claims abstract description 8
- 239000012228 culture supernatant Substances 0.000 claims abstract description 8
- 239000007788 liquid Substances 0.000 claims abstract description 8
- 238000012258 culturing Methods 0.000 claims abstract description 7
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 5
- 229930006000 Sucrose Natural products 0.000 claims abstract description 5
- 239000012452 mother liquor Substances 0.000 claims abstract description 5
- 239000005720 sucrose Substances 0.000 claims abstract description 5
- 239000006228 supernatant Substances 0.000 claims abstract description 5
- 238000009777 vacuum freeze-drying Methods 0.000 claims abstract description 3
- 239000002609 medium Substances 0.000 claims description 22
- 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 claims description 16
- 238000000034 method Methods 0.000 claims description 15
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 claims description 12
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 11
- 238000004113 cell culture Methods 0.000 claims description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 8
- 229960005322 streptomycin Drugs 0.000 claims description 8
- 210000003954 umbilical cord Anatomy 0.000 claims description 8
- 229910002092 carbon dioxide Inorganic materials 0.000 claims description 6
- 235000020776 essential amino acid Nutrition 0.000 claims description 6
- 239000003797 essential amino acid Substances 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 6
- 229940054269 sodium pyruvate Drugs 0.000 claims description 6
- 239000012894 fetal calf serum Substances 0.000 claims description 5
- 229930182555 Penicillin Natural products 0.000 claims description 4
- 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 claims description 4
- 239000001569 carbon dioxide Substances 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 238000007710 freezing Methods 0.000 claims description 4
- 235000015110 jellies Nutrition 0.000 claims description 4
- 239000008274 jelly Substances 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 229940049954 penicillin Drugs 0.000 claims description 4
- 229910052708 sodium Inorganic materials 0.000 claims description 4
- 239000011734 sodium Substances 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 230000001954 sterilising effect Effects 0.000 claims description 4
- 210000004204 blood vessel Anatomy 0.000 claims description 3
- 238000005520 cutting process Methods 0.000 claims description 3
- 230000004927 fusion Effects 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 2
- 229930182816 L-glutamine Natural products 0.000 claims description 2
- 230000001464 adherent effect Effects 0.000 claims description 2
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 claims description 2
- 230000008014 freezing Effects 0.000 claims description 2
- 238000002955 isolation Methods 0.000 claims description 2
- 239000010413 mother solution Substances 0.000 claims description 2
- 238000005303 weighing Methods 0.000 claims description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 2
- 230000008472 epithelial growth Effects 0.000 claims 1
- 239000003102 growth factor Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 9
- 230000010261 cell growth Effects 0.000 description 11
- 102100021669 Stromal cell-derived factor 1 Human genes 0.000 description 10
- 101710088580 Stromal cell-derived factor 1 Proteins 0.000 description 10
- 239000000523 sample Substances 0.000 description 10
- HBBGRARXTFLTSG-UHFFFAOYSA-N Lithium ion Chemical compound [Li+] HBBGRARXTFLTSG-UHFFFAOYSA-N 0.000 description 9
- 230000000975 bioactive effect Effects 0.000 description 7
- 230000006870 function Effects 0.000 description 7
- 230000012010 growth Effects 0.000 description 7
- 229910001416 lithium ion Inorganic materials 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 208000028990 Skin injury Diseases 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 230000001737 promoting effect Effects 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 208000027418 Wounds and injury Diseases 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 230000002500 effect on skin Effects 0.000 description 4
- 210000002950 fibroblast Anatomy 0.000 description 4
- 239000011550 stock solution Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 230000008439 repair process Effects 0.000 description 3
- 230000009759 skin aging Effects 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 208000008960 Diabetic foot Diseases 0.000 description 2
- 102000001267 GSK3 Human genes 0.000 description 2
- 108010014905 Glycogen Synthase Kinase 3 Proteins 0.000 description 2
- 230000032683 aging Effects 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 230000000451 tissue damage Effects 0.000 description 2
- 231100000827 tissue damage Toxicity 0.000 description 2
- 108091032955 Bacterial small RNA Proteins 0.000 description 1
- 102000006573 Chemokine CXCL12 Human genes 0.000 description 1
- 108010008951 Chemokine CXCL12 Proteins 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 108020005199 Dehydrogenases Proteins 0.000 description 1
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 1
- 101800003838 Epidermal growth factor Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 238000000134 MTT assay Methods 0.000 description 1
- 231100000002 MTT assay Toxicity 0.000 description 1
- IXKSXJFAGXLQOQ-XISFHERQSA-N WHWLQLKPGQPMY Chemical compound C([C@@H](C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)C1=CNC=N1 IXKSXJFAGXLQOQ-XISFHERQSA-N 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000002457 bidirectional effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229940116977 epidermal growth factor Drugs 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 210000003953 foreskin Anatomy 0.000 description 1
- 230000037313 granulation tissue formation Effects 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 230000004112 neuroprotection Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000011164 ossification Effects 0.000 description 1
- 239000013630 prepared media Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 108010048734 sclerotin Proteins 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 210000004927 skin cell Anatomy 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
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
- C12N5/0668—Mesenchymal stem cells from other natural sources
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/02—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings containing insect repellants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- 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
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
-
- 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
-
- 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
- C12N2500/44—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
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- 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
- C12N2501/70—Enzymes
- C12N2501/72—Transferases [EC 2.]
- C12N2501/727—Kinases (EC 2.7.)
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biomedical Technology (AREA)
- Dermatology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Developmental Biology & Embryology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Rheumatology (AREA)
- General Engineering & Computer Science (AREA)
- Cell Biology (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Tropical Medicine & Parasitology (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种间充质干细胞培养基用于化妆品的制备方法，包括以下步骤：(1)1号培养基的制备；(2)氯化锂母液的制备；(3)2号培养基的制备；(4)间充质干细胞的分离与培养；(5)间充质干细胞条件培养基的制备：取第四次传代的细胞，将细胞以1x105/ml的密度分别接种于1号培养基和2号培养基中，弃去培养上清，加入适量1X HBSS溶液清洗细胞两次，彻底吸干残留的HBSS溶液，加入无血清的高糖DMEM培养基，去除细胞碎片，向每个上清中加入适量20％无菌蔗糖溶液，将上述液体分装成10ml/瓶，转入真空冷冻干燥瓶中，在真空冷冻干燥机中冻干后保存于‑80℃冰箱中备用。本发明与现有技术相比的优点在于：使用效果好、利用率高、成本低。
Description
技术领域
本发明涉及间充质干细胞技术领域，具体是指一种间充质干细胞培养基用于化妆品的制备方法。
背景技术
间充质干细胞(MSC)是一类各种组织中普遍存在的、与相应的组织损伤的修复有着密切关系的一类异质性细胞，是修复组织器官的主要原料来源之一；MSCs在体内生长过程中，会通过分泌一些细胞因子抑制炎症的发展，并调节形成新生血管，促进血管形成、促进创伤组织的细胞生长，参与创面的损伤修复，最终促进创面上皮化和肉芽组织形成，实现加速创面愈合的进程；体外培养的MSC所分泌的这些具有组织损伤修复功能的生物活性分子，会释放到培养上清中，这种培养上清收集后被称为MSC条件培养基。
MSC的条件培养基(Mesenchymal Stem Cell Conditional Medium，MSC-CM)是指间充质干细胞(MSC) 在体外培养一段时间以后收集的细胞培养上清，其中含有MSC在生长过程中所分泌的具有生物活性的蛋白质分子，如具有调节细胞生长和血管组织生成的细胞因子，核酸分子，如具有细胞生长调节功能的小 RNA(MicroRNA)等生物活性分子。众多的文献报道MSC-CM的成分相对比较复杂，其生物学功能主要为调节细胞生长和分化由于MSC-CM在皮肤损伤的修复与皮肤的抗衰老方面具有较强的功能，MSC-CM 有望应用于皮肤损伤的修复(如烧伤、糖尿病足等)和皮肤抗衰老为目的的化妆品中，吸引了众多的研究者开发新的方法与技术，提高MSC-CM的产量，增强其生物学功能，降低产品制备的成本。
研究表明锂离子是一种双向调节的化合物，实验动物的研究表明适量的锂元素有助于骨质的形成，同时适量的锂元素对于神经细胞也具有保护作用，培养基中低浓度的锂离子可以促进MSC的增殖，而高浓度的锂离子则会抑制MSC的增殖与分化。普遍认为，锂离子有助于骨质形成与神经保护作用是由于锂离子促进了MSC的增殖和分化，而对于是否锂离子的使用使MSC所分泌的活性因子有所增加，改善了细胞生长的微环境，目前未见报道。在我们的研究中发现在培养基中添加低浓度的氯化锂将有助于提高 MSC-CM中活性物质的产量。
现有技术中的间充质干细胞培养基用于化妆品的制备方法在使用时，不仅使用效果不好，间充质干细胞条件培养基中含有活细胞，在使用细胞时具有免疫排斥、潜在的致瘤性、不易保存与运输等缺点，而且间充质干细胞的利用率低，提高了间充质干细胞条件培养基的制备成本。
发明内容
本发明要解决的技术问题是克服以上技术缺陷，提供一种使用效果好、利用率高、成本低的一种间充质干细胞培养基用于化妆品的制备方法。
为解决上述技术问题，本发明提供的技术方案为：一种间充质干细胞培养基用于化妆品的制备方法，包括以下步骤：
(1)1号培养基的制备：取420ml的高糖MEM倒入容器中，向其中分别加入5ml 100X青/链霉素混合液、5ml 100X丙酮酸钠、5ml 100X非必须氨基酸、5ml 100X谷氨酰钠和50ml胎牛血清，混匀后，过滤除菌，备用；
(2)氯化锂母液的制备：称取100mg氯化锂固体，溶于8ml高糖MEM中，充分溶解后定容至10ml，此溶液浓度为10mg/ml；
(3)2号培养基的制备：取416ml的高糖MEM倒入容器中，向其中分别加入5ml 100X青/链霉素混合液、5ml 100X丙酮酸钠、5ml 100X非必须氨基酸、5ml 100X谷氨酰钠和50ml胎牛血清，同时添加氯化锂母液4ml，混匀后，过滤除菌，备用；
(4)间充质干细胞(MSC)的分离与培养：将脐带组织沿脐带纵向切开，剥离其中的血管；用剪刀分离脐带组织内侧的沃顿胶，将分离的沃顿胶切成1mm3的小块，然后将其接种于细胞培养皿中，加入适量的1号培养液，将培养皿放置于温度为37℃、二氧化碳浓度为5％的培养箱中培养10-14天，期间于第二天适当加液后，每隔三天换1号培养液一次；细胞培养至80％融合度，用0.5％胰酶-EDTA溶液消化已经贴壁的间充质干细胞，并按照1：3的比例进行传代培养，传代后的细胞取其中一部分进行间充质干细胞条件培养基的制备，其余细胞以2x106/ml的密度进行冻存；
(5)间充质干细胞条件培养基(MSC-CM)的制备：取第四次传代的细胞，将细胞以1x105/ml的密度分别接种于1号培养基和2号培养基中，待细胞在两种培养基中生长至90％融合度，小心弃去培养上清，加入适量1X HBSS溶液(对于10cm细胞培养皿，可加入10ml-15ml HBSS溶液)清洗细胞两次，彻底吸干残留的HBSS溶液，加入无血清的高糖DMEM培养基，于温度为37℃、二氧化碳浓度为5％的培养箱中继续培养72h后，将2种细胞培养上清分别收集于50ml离心管中，1000g离心10分钟，去除细胞碎片，测量上清体积，向每个上清中加入适量20％无菌蔗糖溶液，使蔗糖的终浓度达到1％(W/V)，将上述液体分装成10ml/瓶(青霉素瓶)，转入真空冷冻干燥瓶中，-80℃预冷冻后，在真空冷冻干燥机中冻干后保存于-80℃冰箱中备用。两种条件培养基分别标记为MSC-CM 1：在1号培养基中培养的MSC所制备的条件培养基；MSC-CM2：2号培养基(含氯化锂的培养基)中培养的MSC所制备的条件培养基。
本发明与现有技术相比的优点在于：本发明利用在MSC体外培养的无血清培养基中添加GSK3 (Glycogen Synthase Kinase 3)小分子抑制剂：锂离子，用以降低MSC在体外培养过程中发生的细胞分化，提高其所分泌的生物活性分子的分泌能力，提高MSC-CM中有效成分的产量，在最佳的使用条件下， MSC-CM促进人真皮成纤维细胞生长的滴度提高了5-10倍，不仅间充质干细胞条件培养基中不含有活细胞，在使用细胞时没有免疫排斥、潜在的致瘤性、不易保存与运输等缺点，提高了使用效果，而且提高了间充质干细胞的利用，降低了间充质干细胞条件培养基的制备成本；利用含有本药物的化妆品可用于皮肤损伤及老化的修复，所指的皮肤损伤包括但不限于外伤造成的皮肤损伤、慢性疾病造成的皮肤损伤(如糖尿病足等)以及年龄增长所造成的皮肤老化等现象进行修复，提高了使用效果。
作为改进，所述(1)中青/链霉素混合液的终浓度为100ug/ml。
作为改进，所述(5)中的高糖DMEM含青霉素(100u/ml)/链霉素(100ug/ml)，并添加以下相应成分：丙酮酸钠(1mM)、非必须氨基酸(10uM)、L-谷氨酰胺(2mM)、巯基乙醇(55uM)、上表皮生长因素(10ng/ml)和氯化锂母液(80ug/ml)。
作为改进，所述1号培养基和2号培养基的容积为500ml。
作为改进，一种间充质干细胞条件培养基，其通过权利要求1-4中任一项所述的方法制备。
附图说明
图1是本发明一种间充质干细胞培养基用于化妆品的制备方法的脐带间充质干细胞(MSC)表面标志物的流式鉴定结果图。
图2是本发明一种间充质干细胞培养基用于化妆品的制备方法的SDF-1α标准曲线的测定图。
图3是本发明一种间充质干细胞培养基用于化妆品的制备方法的MTT检测不同方式制备的条件培养基对细胞生长的作用结果图。
具体实施方式
下面通过具体实施例对本发明作进一步详述，以下实施例只是描述性的，不是限定性的，不能一次限定本发明的保护范围。
实施例1间充质干细胞(MSC)的表面标志物鉴定
第四次传代(P4)的体外培养的MSC冻存前取3x106个细胞，DPBS清洗后，800g离心5分钟，重悬于300ul的含1％牛血清白蛋白(BSA)的DPBS中，平均分成三份，其中1份加入流式检测的同型对照抗体，另外2份分别加入两组流式检测抗体：1个样品中加入FITC-HLA-DR、APC-CD90、PE-CD105和 Percp-CD34，另外1个样品中加入PE-CD73和Percp-CD45RA，4℃染色30min后，用含1％BSA的DPBS 清洗1遍，800g离心5分钟，上机检测，流式仪的型号为BD-FacsCalibur。流式细胞仪检测结果用Flowjo 软件进行分析，在FSCvsSSC图中选取细胞部分，使用Histogram检测MSC表面标志物的表达情况。结果表明检测的MSC中CD105、CD90和CD73均为阳性表达，阳性细胞的比例均超过90％，而HLA-DR、 CD45RA的表达均为阴性，结果见附图1。
实施例2MSC-CM中重要生物活性物质的检测(ELISA)
以MSC培养中最为常见的因子SDF-1α为对象，用定量ELISA方法测定MSC-CM中SDF-1α因子的测定含量，ELISA试剂盒使用R&D System公司，(目录号：DSA00)，检测过程严格按照试剂盒生产厂家所提供的说明书进行，主要步骤简述如下：
①检测样品的准备：2种方法制备的冻干MSC-CM各取一支，分别用0.5ml无菌、无热源的去离子水溶解，取100ul溶解后的MSC-CM，加入900ul试剂盒提供的样品稀释液，将样品稀释10倍，然后取200ul 10倍稀释后的样品，用试剂盒提供的样品稀释液继续稀释2倍，备用；
②SDF-1α标准品的制备：按照说明书要求将试剂盒提供的SDF-1α的标准品(100ug/ml)用样品稀释液稀释后得到浓度分别为10000pg/ml、5000pg/ml、2500pg/ml、1250pg/ml、625pg/ml、313pg/ml 和156pg/ml的系列标准品；
③向试剂盒中提供的ELISA板中加入100ul/孔的样品稀释液，然后加入100ul/孔上述制备的20倍、 40倍和80倍稀释的MSC-CM和SDF-1α标准品，置水平摇床500rpm，室温孵育2小时；检测样品和标准品均设置平行孔。
④孵育结束后，吸去孵育液，加入试剂盒提供的洗液400ul/孔，洗涤3次，吸干洗液后，加入200ul/ 孔SDF-1α结合物，500rpm水平摇床室温孵育2小时；
⑤重复步骤③中的洗涤步骤，彻底吸干残留洗液；
⑥加入200ul/孔底物显色液，室温、避光反应30分钟后，每孔加入50ul终止液，于450nm波长(使用570nm作为校正波长)读取结果。
⑦根据所测OD值，结合标准品的标准曲线(表1)，确定样品中的SDF-1α的含量(表2)。
⑧检测结果：见附图2。
表1 SDF-1alpha测定标准曲线的测定
两个样品MSC-CM1和MSC-CM2测定的OD值及根据标准曲线线性回归公式所计算的SDF-1α在相应样品中的含量见下表：
表2 SDF-1α在相应样品中的含量
实施例3 MSC-CM对上皮细胞生长的影响的检测
MSC-CM中生物活性分子的功能通过检测MSC-CM对人真皮成纤维细胞生长的影响来进行测定，人真皮成纤维细胞采用HDF(Human dermal fiborblast，人真皮成纤维细胞，购自美国Cell Applications(Cat#： 106K-05a)，为16周岁人包皮细胞分离而来)。MTT试剂盒购自碧云天生物技术有限公司，产品目录号：C0009。
①测试样品培养基的制备：首先配制500ml含10％胎牛血清的DMEM/F12培养基，备用(此培养基也是HDF细胞生长培养基)，取2种不同方式制备的MSC-CM冻干品，每支冻干品中加入1ml上述培养基使其充分溶解，(此培养基为MSC-CM培养基原液)，然后用上述配制的10％FCS的DMEM/F12培养基稀释MSC-CM1 和MSC-CM2，制备5倍和10倍的MSC-CM1和MSC-CM2的稀释培养基。
②培养的HDF细胞达到80～90％融合后，0.5％胰酶-EDTA消化，消化后的细胞用10％FCS DMEM/F12 培养基以5×104/ml重悬细胞，并将其接种于96孔培养板中，每孔100μl，37℃5％CO2细胞培养箱内培养24小时；
③24h后弃原培养液，各组分别加入100ul步骤①配制的MSC-CM1和MSC-CM2原液、5X和10X培养基，每个MSC-CM设3个平行孔，同时设空白培养基和普通培养基孔为实验对照。放入37℃5％CO2培养箱中分别培养24h、48h、72h。
④各观察期终止时，按照试剂盒说明书加入10μL/孔MTT液，继续培养4h，吸去原液，加入100 μL/孔产物溶解液。37度孵育4小时，取出震荡10min，在酶标仪上以570nm波长测定吸光度。由于MTT 可以被活细胞内的线粒体中的一些脱氢酶还原生成结晶状的深紫色产物formazan。在特定溶剂(洗涤剂或 DMSO)存在的情况下，可以被完全溶解。然后通过酶标仪可以测定570nm波长附近的吸光度。细胞增殖越多越快，则吸光度越高；本实验重复3次。
⑤检测结果：本实验检测了不同稀释度的条件培养基样品对人真皮成纤维细胞(HDF)生长情况的影响，以此来说明条件培养基中的生物活性分子具有促进人皮肤细胞的生长的潜能，为其未来的应用指明方向。实验结果表明，在添加有MSC-CM原液的细胞培养板中，经过3天的测试，细胞生长的速度明显高于对照培养基(P<0.001)；随着两种MSC-CM稀释度的提高，其促进细胞增殖的功能逐步减弱，其中CM1 的促进作用在5倍稀释后基本与对照培养基的生长速度相当，而CM2在10倍稀释后所配制的培养基中仍具有促进细胞生长的能力，结果见附图3。
Claims (5)
1.一种间充质干细胞培养基用于化妆品的制备方法，其特征在于，包括以下步骤：
(1)1号培养基的制备：取420ml的高糖MEM倒入容器中，向其中分别加入5ml 100X青/链霉素混合液、5ml 100X丙酮酸钠、5ml 100X非必须氨基酸、5ml 100X谷氨酰钠和50ml胎牛血清，混匀后，过滤除菌，备用；
(2)氯化锂母液的制备：称取100mg氯化锂固体，溶于8ml高糖MEM中，充分溶解后定容至10ml，此溶液浓度为10mg/ml；
(3)2号培养基的制备：取416ml的高糖MEM倒入容器中，向其中分别加入5ml 100X青/链霉素混合液、5ml 100X丙酮酸钠、5ml 100X非必须氨基酸、5ml 100X谷氨酰钠和50ml胎牛血清，同时添加氯化锂母液4ml，混匀后，过滤除菌，备用；
(4)间充质干细胞(MSC)的分离与培养：将脐带组织沿脐带纵向切开，剥离其中的血管；用剪刀分离脐带组织内侧的沃顿胶，将分离的沃顿胶切成1mm3的小块，然后将其接种于细胞培养皿中，加入适量的1号培养液，将培养皿放置于温度为37℃、二氧化碳浓度为5％的培养箱中培养10-14天，期间于第二天适当加液后，每隔三天换1号培养液一次；细胞培养至80％融合度，用0.5％胰酶-EDTA溶液消化已经贴壁的间充质干细胞，并按照1：3的比例进行传代培养，传代后的细胞取其中一部分进行间充质干细胞条件培养基的制备，其余细胞以2x106/ml的密度进行冻存；
(5)间充质干细胞条件培养基(MSC-CM)的制备：取第四次传代的细胞，将细胞以1x105/ml的密度分别接种于1号培养基和2号培养基中，待细胞在两种培养基中生长至90％融合度，小心弃去培养上清，加入适量1X HBSS溶液(对于10cm细胞培养皿，可加入10ml-15mlHBSS溶液)清洗细胞两次，彻底吸干残留的HBSS溶液，加入无血清的高糖DMEM培养基，于温度为37℃、二氧化碳浓度为5％的培养箱中继续培养72h后，将2种细胞培养上清分别收集于50ml离心管中，1000g离心10分钟，去除细胞碎片，测量上清体积，向每个上清中加入适量20％无菌蔗糖溶液，使蔗糖的终浓度达到1％(W/V)，将上述液体分装成10ml/瓶(青霉素瓶)，转入真空冷冻干燥瓶中，-80℃预冷冻后，在真空冷冻干燥机中冻干后保存于-80℃冰箱中备用。两种条件培养基分别标记为MSC-CM 1：在1号培养基中培养的MSC所制备的条件培养基；MSC-CM2：2号培养基(含氯化锂的培养基)中培养的MSC所制备的条件培养基。
2.根据权利要求1所述的一种间充质干细胞培养基用于化妆品的制备方法，其特征在于：所述(1)中青/链霉素混合液的终浓度为100ug/ml。
3.根据权利要求1所述的一种间充质干细胞培养基用于化妆品的制备方法，其特征在于：所述(5)中的高糖DMEM含青霉素(100u/ml)/链霉素(100ug/ml)，并添加以下相应成分：丙酮酸钠(1mM)、非必须氨基酸(10uM)、L-谷氨酰胺(2mM)、巯基乙醇(55uM)、上表皮生长因素(10ng/ml)和氯化锂母液(80ug/ml)。
4.根据权利要求1所述的一种间充质干细胞培养基用于化妆品的制备方法，其特征在于：所述1号培养基和2号培养基的容积为500ml。
5.一种间充质干细胞条件培养基，其通过权利要求1-4中任一项所述的方法制备。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010328316.9A CN111454898A (zh) | 2020-04-23 | 2020-04-23 | 一种间充质干细胞培养基用于化妆品的制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010328316.9A CN111454898A (zh) | 2020-04-23 | 2020-04-23 | 一种间充质干细胞培养基用于化妆品的制备方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111454898A true CN111454898A (zh) | 2020-07-28 |
Family
ID=71676874
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010328316.9A Pending CN111454898A (zh) | 2020-04-23 | 2020-04-23 | 一种间充质干细胞培养基用于化妆品的制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111454898A (zh) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100189696A1 (en) * | 2006-11-01 | 2010-07-29 | Dongming Sun | Lithium stimulation of cord blood stem cell proliferation and growth factor production |
| US20100279412A1 (en) * | 2006-01-13 | 2010-11-04 | Japan Science And Technology Agency | Culture medium additive for use in serum-free culturing of animal cell, kit and use thereof |
| CN104997708A (zh) * | 2015-07-07 | 2015-10-28 | 河南中科干细胞基因工程有限公司 | 干细胞条件培养基的获取及在防皮肤老化方面的应用 |
| CN106318904A (zh) * | 2015-06-15 | 2017-01-11 | 上海朗斯生物工程有限公司 | 间充质干细胞条件培养基用于美容品领域的用途 |
| CN107779430A (zh) * | 2016-08-30 | 2018-03-09 | 天津市康婷生物工程有限公司 | 脐带间充质干细胞上清液的收集方法 |
-
2020
- 2020-04-23 CN CN202010328316.9A patent/CN111454898A/zh active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100279412A1 (en) * | 2006-01-13 | 2010-11-04 | Japan Science And Technology Agency | Culture medium additive for use in serum-free culturing of animal cell, kit and use thereof |
| US20100189696A1 (en) * | 2006-11-01 | 2010-07-29 | Dongming Sun | Lithium stimulation of cord blood stem cell proliferation and growth factor production |
| CN106318904A (zh) * | 2015-06-15 | 2017-01-11 | 上海朗斯生物工程有限公司 | 间充质干细胞条件培养基用于美容品领域的用途 |
| CN104997708A (zh) * | 2015-07-07 | 2015-10-28 | 河南中科干细胞基因工程有限公司 | 干细胞条件培养基的获取及在防皮肤老化方面的应用 |
| CN107779430A (zh) * | 2016-08-30 | 2018-03-09 | 天津市康婷生物工程有限公司 | 脐带间充质干细胞上清液的收集方法 |
Non-Patent Citations (1)
| Title |
|---|
| YOON-JIN KIM等: "Exosomes derived from human umbilical cord blood mesenchymal stem cells stimulates rejuvenation of human skin", 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104673747B (zh) | 一种血小板裂解液的制备方法及其应用 | |
| AU2017339344B2 (en) | A method of isolating mesenchymal stem cells from umbilical cord amniotic membrane using a cell culture medium | |
| CN107746829B (zh) | 一种分离与原代培养犬胎盘来源的间充质干细胞的方法 | |
| AU2019250653A1 (en) | A method of inducing or improving wound healing properties of mesenchymal stem cells | |
| CN114642630B (zh) | 一种负载牙龈间充质干细胞外泌体的矿化胶原凝胶及其制备方法 | |
| CN111454898A (zh) | 一种间充质干细胞培养基用于化妆品的制备方法 | |
| CN1597937A (zh) | 胎盘羊膜细胞提取物及其在间充质干细胞诱导分化中的应用 | |
| Li et al. | Method of culturing cardiomyocytes from human pediatric ventricular myocardium | |
| CN108823154A (zh) | 一种脂肪干细胞的无血清培养基及其制备方法 | |
| CN111808807A (zh) | 一种无需预先包被的间充质干细胞无血清培养基及其应用 | |
| RU2384618C2 (ru) | Способ получения фибробластоподобных клеток из пупочного канатика новорожденного | |
| CN102041243A (zh) | 一种快速分离骨髓间充质干细胞的试剂盒及方法 | |
| JP7002497B2 (ja) | シート状細胞培養物の製造方法 | |
| US20210301252A9 (en) | Method of inducing or improving wound healing properties of mesenchymal stem cells | |
| Erfanmanesh et al. | Comparison of the effect of bovine platelet lysate and platelet rich plasma as growth promoters on growth rate and viability of different cell lines. | |
| CN111000791A (zh) | 含有脂肪干细胞外泌体的面膜的制备方法 | |
| CN111394301A (zh) | 白皮杉醇在增加多能干细胞分泌外泌体数量及生物活性中的应用 | |
| 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 | |
| CN115433704B (zh) | 一种细胞消化液以及利用该细胞消化液对口腔黏膜上皮细胞分离培养的方法 | |
| CN112662621B (zh) | 一种逆转间充质干细胞衰老的方法及应用 | |
| CN113308438B (zh) | Fto基因修饰的猪脂肪干细胞及其构建方法与应用 | |
| CN116693599A (zh) | 制备富血小板纤维蛋白提取液的方法及采用PRFe培养牙髓干细胞的方法 | |
| CN105497069A (zh) | 用于皮肤护理的组合物及其医药组合与其制备方法 | |
| RU2620342C1 (ru) | Косметическое средство на основе продуктов секреции мезенхимных стволовых/стромальных клеток жировой ткани человека и способ его получения | |
| CN118389419A (zh) | 一种经血间充质干细胞的提取培养方法 |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200728 |
|
| RJ01 | Rejection of invention patent application after publication |