CN107267441B - 一种建立用于抗炎抗敏功效评价的三维皮肤模型的方法 - Google Patents
一种建立用于抗炎抗敏功效评价的三维皮肤模型的方法 Download PDFInfo
- Publication number
- CN107267441B CN107267441B CN201710458682.4A CN201710458682A CN107267441B CN 107267441 B CN107267441 B CN 107267441B CN 201710458682 A CN201710458682 A CN 201710458682A CN 107267441 B CN107267441 B CN 107267441B
- Authority
- CN
- China
- Prior art keywords
- cells
- culturing
- culture
- solution
- days
- 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.)
- Active
Links
- 238000000034 method Methods 0.000 title claims abstract description 38
- 230000003110 anti-inflammatory effect Effects 0.000 title claims abstract description 20
- 230000003266 anti-allergic effect Effects 0.000 title abstract description 15
- 238000011156 evaluation Methods 0.000 title description 8
- 210000003491 skin Anatomy 0.000 claims abstract description 96
- 238000012258 culturing Methods 0.000 claims abstract description 54
- 239000000017 hydrogel Substances 0.000 claims abstract description 32
- 210000002950 fibroblast Anatomy 0.000 claims abstract description 24
- 210000002510 keratinocyte Anatomy 0.000 claims abstract description 22
- 239000007788 liquid Substances 0.000 claims abstract description 15
- 238000012216 screening Methods 0.000 claims abstract description 15
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 claims abstract description 12
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 claims abstract description 12
- 210000002744 extracellular matrix Anatomy 0.000 claims abstract description 12
- 229920000642 polymer Polymers 0.000 claims abstract description 11
- 210000002615 epidermis Anatomy 0.000 claims abstract description 9
- 238000002156 mixing Methods 0.000 claims abstract description 8
- 210000004207 dermis Anatomy 0.000 claims abstract description 6
- 239000000203 mixture Substances 0.000 claims abstract description 3
- 210000004027 cell Anatomy 0.000 claims description 48
- 239000000243 solution Substances 0.000 claims description 48
- 239000001963 growth medium Substances 0.000 claims description 29
- 210000005087 mononuclear cell Anatomy 0.000 claims description 25
- 239000012980 RPMI-1640 medium Substances 0.000 claims description 24
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 23
- 210000001519 tissue Anatomy 0.000 claims description 16
- 210000003630 histaminocyte Anatomy 0.000 claims description 15
- 210000002865 immune cell Anatomy 0.000 claims description 15
- 210000004698 lymphocyte Anatomy 0.000 claims description 15
- 239000002609 medium Substances 0.000 claims description 14
- 210000004369 blood Anatomy 0.000 claims description 13
- 239000008280 blood Substances 0.000 claims description 13
- 210000003714 granulocyte Anatomy 0.000 claims description 13
- 210000004443 dendritic cell Anatomy 0.000 claims description 11
- 210000002540 macrophage Anatomy 0.000 claims description 11
- 210000001616 monocyte Anatomy 0.000 claims description 11
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 10
- 102000012422 Collagen Type I Human genes 0.000 claims description 9
- 108010022452 Collagen Type I Proteins 0.000 claims description 9
- 239000002202 Polyethylene glycol Substances 0.000 claims description 8
- 125000004386 diacrylate group Chemical group 0.000 claims description 8
- 229920001223 polyethylene glycol Polymers 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 7
- 238000005520 cutting process Methods 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- 230000001464 adherent effect Effects 0.000 claims description 6
- 239000000706 filtrate Substances 0.000 claims description 6
- 229920002674 hyaluronan Polymers 0.000 claims description 6
- 229960003160 hyaluronic acid Drugs 0.000 claims description 6
- PHEDXBVPIONUQT-RGYGYFBISA-N phorbol 13-acetate 12-myristate Chemical compound C([C@]1(O)C(=O)C(C)=C[C@H]1[C@@]1(O)[C@H](C)[C@H]2OC(=O)CCCCCCCCCCCCC)C(CO)=C[C@H]1[C@H]1[C@]2(OC(C)=O)C1(C)C PHEDXBVPIONUQT-RGYGYFBISA-N 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 102000029816 Collagenase Human genes 0.000 claims description 5
- 108060005980 Collagenase Proteins 0.000 claims description 5
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 claims description 5
- 108010003272 Hyaluronate lyase Proteins 0.000 claims description 5
- 102000001974 Hyaluronidases Human genes 0.000 claims description 5
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 claims description 5
- 239000000043 antiallergic agent Substances 0.000 claims description 5
- 229960002424 collagenase Drugs 0.000 claims description 5
- 230000001079 digestive effect Effects 0.000 claims description 5
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 5
- 229960002773 hyaluronidase Drugs 0.000 claims description 5
- 230000002500 effect on skin Effects 0.000 claims description 4
- 238000000684 flow cytometry Methods 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- 108090000978 Interleukin-4 Proteins 0.000 claims description 3
- 239000011324 bead Substances 0.000 claims description 3
- 230000024245 cell differentiation Effects 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000002644 phorbol ester Substances 0.000 claims description 3
- 231100000241 scar Toxicity 0.000 claims description 3
- 238000007790 scraping Methods 0.000 claims description 3
- 238000010008 shearing Methods 0.000 claims description 3
- 210000004003 subcutaneous fat Anatomy 0.000 claims description 3
- 230000029087 digestion Effects 0.000 claims description 2
- 238000012545 processing Methods 0.000 claims description 2
- 239000003814 drug Substances 0.000 abstract description 7
- 238000002360 preparation method Methods 0.000 abstract description 4
- 239000012188 paraffin wax Substances 0.000 description 11
- 239000000126 substance Substances 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 8
- 238000000338 in vitro Methods 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 7
- 210000005259 peripheral blood Anatomy 0.000 description 7
- 239000011886 peripheral blood Substances 0.000 description 7
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 239000008055 phosphate buffer solution Substances 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 239000002158 endotoxin Substances 0.000 description 5
- 230000004054 inflammatory process Effects 0.000 description 5
- 229920006008 lipopolysaccharide Polymers 0.000 description 5
- 206010061218 Inflammation Diseases 0.000 description 4
- 239000002537 cosmetic Substances 0.000 description 4
- 238000012136 culture method Methods 0.000 description 4
- 230000002757 inflammatory effect Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- ZHHYXNZJDGDGPJ-BSWSSELBSA-N (2e,4e)-nona-2,4-dienal Chemical compound CCCC\C=C\C=C\C=O ZHHYXNZJDGDGPJ-BSWSSELBSA-N 0.000 description 3
- ZHHYXNZJDGDGPJ-UHFFFAOYSA-N 2,4-Nonadienal Natural products CCCCC=CC=CC=O ZHHYXNZJDGDGPJ-UHFFFAOYSA-N 0.000 description 3
- 201000004624 Dermatitis Diseases 0.000 description 3
- ZFMITUMMTDLWHR-UHFFFAOYSA-N Minoxidil Chemical compound NC1=[N+]([O-])C(N)=CC(N2CCCCC2)=N1 ZFMITUMMTDLWHR-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 229960003632 minoxidil Drugs 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 241001272567 Hominoidea Species 0.000 description 2
- 206010070834 Sensitisation Diseases 0.000 description 2
- 206010070835 Skin sensitisation Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 208000010668 atopic eczema Diseases 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000008313 sensitization Effects 0.000 description 2
- 238000004088 simulation Methods 0.000 description 2
- 230000037380 skin damage Effects 0.000 description 2
- 231100000370 skin sensitisation Toxicity 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- -1 sulfhydryl modified hyaluronic acid Chemical class 0.000 description 2
- 229950003937 tolonium Drugs 0.000 description 2
- HNONEKILPDHFOL-UHFFFAOYSA-M tolonium chloride Chemical compound [Cl-].C1=C(C)C(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 HNONEKILPDHFOL-UHFFFAOYSA-M 0.000 description 2
- HKEBYUNPANBGPL-WJDMQLPWSA-N (2e,4e)-nona-2,4-diene Chemical compound CCCC\C=C\C=C\C HKEBYUNPANBGPL-WJDMQLPWSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 208000027205 Congenital disease Diseases 0.000 description 1
- 206010014025 Ear swelling Diseases 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 235000009161 Espostoa lanata Nutrition 0.000 description 1
- 240000001624 Espostoa lanata Species 0.000 description 1
- 206010018691 Granuloma Diseases 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 101001046686 Homo sapiens Integrin alpha-M Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 102100022338 Integrin alpha-M Human genes 0.000 description 1
- 102100022297 Integrin alpha-X Human genes 0.000 description 1
- 240000001307 Myosotis scorpioides Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- XKRFYHLGVUSROY-UHFFFAOYSA-N argon Substances [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000001045 blue dye Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000002458 cell surface marker Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000005857 detection of stimulus Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 210000005175 epidermal keratinocyte Anatomy 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 230000007773 growth pattern Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 230000037311 normal skin Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000004976 peripheral blood cell Anatomy 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001012 protector Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000009759 skin aging Effects 0.000 description 1
- 208000017520 skin disease Diseases 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 239000008096 xylene Substances 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
- C12N5/0656—Adult fibroblasts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
-
- 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
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0629—Keratinocytes; Whole skin
-
- 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
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
-
- 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
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
-
- 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
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0642—Granulocytes, e.g. basopils, eosinophils, neutrophils, mast cells
-
- 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
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
-
- 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
- C12N2513/00—3D culture
-
- 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
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/30—Synthetic polymers
-
- 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
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
-
- 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
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/70—Polysaccharides
- C12N2533/80—Hyaluronan
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Rheumatology (AREA)
- Gastroenterology & Hepatology (AREA)
- Pathology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Urology & Nephrology (AREA)
- Toxicology (AREA)
- Endocrinology (AREA)
- Diabetes (AREA)
- Dermatology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明一种三维皮肤模型的建立方法,包括如下步骤:1)制备细胞外基质高分子水凝胶溶液;2)将成纤维细胞与细胞外基质高分子水凝胶溶液混合,放入培养小室中培养;3)接种角质形成细胞,通过气‑液界面培养,得到包含真皮层、表皮层和角质层的三维皮肤模型。本发明方法可以有效建立三维皮肤模型,而且制备方法简便,可以用于皮肤抗炎抗敏药物的筛选,应用前景优良。
Description
技术领域
本发明涉及皮肤模型的建立方法,具体地,涉及用于建立抗炎抗敏功效评价的新型三维皮肤模型的方法。
背景技术
皮肤作为覆盖和保护体表的重要组织器官,阻挡外界物质干扰的第一道关卡,容易受到外伤、烧伤、炎症、溃疡、肿瘤术后及先天疾病等因素的损害,同时会通过某些特殊反应进行防御。全世界皮炎、湿疹等过敏性皮肤病的发病率超过10%。目前在进行皮肤抗炎抗敏药物或化妆品原料的筛选非常不方便,成本也很高,二甲苯致小鼠耳肿胀、蛋清致大鼠足跖肿胀、大鼠棉球肉芽肿慢性炎症模型、小鼠局部***致敏实验是常用的动物实验模型。然而,随着欧洲的化妆品法规颁布关于动物实验在化妆产品中的禁令,让抗炎抗敏的化学品和化妆品原料的筛选和开发更难实现。同时,由于皮肤致敏和炎症机制的复杂性,同一个体外测试***中很难全面反映整个致敏致炎过程,因此,需要开发更多体外实验为化学品和个人护理产品的皮肤抗炎抗敏功效提供预测和评估。
发明内容
为了解决前述问题,本发明的技术方案是提供了一种三维皮肤模型的构建方法及其应用。
本发明三维皮肤模型的建立方法,包括如下步骤:
1)制备细胞外基质高分子水凝胶溶液;
2)将成纤维细胞与细胞外基质高分子水凝胶溶液混合,放入培养小室中培养;
3)接种角质形成细胞,通过气-液界面培养,得到包含真皮层、表皮层和角质层的三维皮肤模型。
步骤1)中,所述细胞外基质高分子水凝胶溶液是透明质酸、聚乙二醇二丙烯酸酯和I型胶原的混合溶液,其中,透明质酸、聚乙二醇二丙烯酸酯和I型胶原的重量比为4:1:1,优选地,它们的浓度分别为6.67mg/ml、1.67mg/ml、1.67mg/ml。
步骤2)中,将成纤维细胞与细胞外基质高分子水凝胶溶液混合的同时,加入1.25×105~2×105个肥大细胞,混合;
优选地,所述肥大细胞按照如下方法制备:取瘢痕组织,除去皮下脂肪,切成小块,加入消化液消化4h,再剪切至乳白色悬液,过滤收集滤液;在4℃条件下离心滤液,弃上清液,重悬,再离心,弃上清液,将悬浮细胞接种入含10%FBS的DMEM培养液中培养,即得肥大细胞;所述消化液是Ⅰ型胶原酶和透明质酸酶的混合溶液,其中Ⅰ型胶原酶的浓度为0.3mg/ml,透明质酸酶的浓度为100U/ml。
步骤2)中,每1ml水凝胶溶液加入5×105~8×105个成纤维细胞,放入培养小室中,置于培养板中培养,培养30min后形成水凝胶,培养的时间为5日。
步骤3)中,接种的角质形成细胞的量为:步骤2)中采用12孔板培养时,每孔加入2×105~4×105角质形成细胞。
步骤3)中,气-液界面培养的方法是:
接种角质形成细胞后,在DMEM培养基淹没培养小室的情况下培养5日,然后在DMEM培养基处于培养小室底部的情况下培养14日;
或者接种角质形成细胞后,先在DMEM培养基淹没培养小室的情况下培养1日,加入免疫细胞至培养小室的周围,继续培养4日,然后在DMEM培养基处于培养小室底部的情况下培养14日,即制备得到;其中,接种的角质形成细胞的量为:步骤2)中采用12孔板培养时,每孔加入2×105~5×105免疫细胞,所述免疫细胞为单核细胞、树突状细胞、T淋巴细胞或者巨噬细胞。
所述成纤维细胞按照如下方法制备:取皮肤真皮层组织,切成小块,采用含有10%FBS的DMEM培养基培养,其间每2天更换一次培养液,培养6天后,回收细胞,通过流式细胞仪对细胞筛选成纤维细胞,即可;
所述角质形成细胞按照如下方法制备:取皮肤表皮层组织,切成小块,采用含有10%FBS的DMEM培养基培养,其间每2天更换一次培养液,培养6天后,回收细胞,通过流式细胞仪对细胞筛选角质形成细胞,即可;
所述单核细胞按照如下方法制备:取血液,采用淋巴细胞分层液处理,离心,取单个核细胞层,采用RPMI-1640培养基,贴壁2h后,去除未贴壁或者未贴牢的细胞,刮下的贴壁细胞为单核细胞;
所述粒细胞按照如下方法制备:取血液,采用淋巴细胞分层液处理,离心,取粒细胞层,洗涤,采用RPMI-1640培养基培养,即得;
所述树突状细胞按照如下方法制备:取血液,采用淋巴细胞分层液处理,离心,取单个核细胞层,采用RPMI-1640培养基,贴壁2h后,去除未贴壁或者未贴牢的细胞,收集贴壁细胞,免疫磁珠法分离CD14+单核细胞,用含有10%FBS、50ng/mL rhGM-CSF和25ng/mL IL-4的RPMI-1640培养液置于37℃、5%CO2培养箱中培养5-6d,得树突状细胞;
所述T淋巴细胞按照如下方法制备:取血液,采用淋巴细胞分层液处理,离心,取单个核细胞层,采用RPMI-1640培养基,贴壁2h后,收集未贴壁的细胞,加入含10%FBS的RPMI-1640培养液,即得T淋巴细胞;
所述巨噬细胞按照如下方法制备:取血液,采用淋巴细胞分层液处理,离心,取单个核细胞层,采用RPMI-1640培养基培养1-2d,加入终浓度为125ng/mL的佛波酯(PMA)的RPMI-1640培养基进行细胞分化,培养2-3d,即得巨噬细胞。
本发明还提供了前述方法建立的三维皮肤模型。
本发明还提供了前述三维皮肤模型在筛选皮肤抗炎抗敏药物中的用途。
本发明还提供了一种筛选治疗皮肤抗炎抗敏药物的方法,包括如下步骤:
a、按照前述的方法建立的三维皮肤模型;
b、将候选物施用于a步骤所述的三维皮肤模型;
c、用三维皮肤模型评价潜在的抗炎抗敏药物。
建立基于皮肤致敏发炎实验整合测试体系提供整合检测策略,研发含有免疫细胞用于抗炎抗敏功效检测的皮肤模型。应用已经构建的皮肤模型建立一个规范化、安全、高效的体外护肤保健品三维皮肤评价体系,作为替代传统动物实验的方法之一,对单个化学品与产品安全性及功效性检测的体外评价方法,验证其实验稳定性、可重复性、以及作为替代方法的可行性,为三维皮肤研究提供重要参考,为化学品和个人护理产品的皮肤抗敏抗炎功效提供预测和评估,同时为我国在替代动物实验法方面提供具有参考意义的依据。
三维皮肤(3D-Skin)是一种较为完善的组织工程化皮肤,是将真皮成纤维细胞、表皮角质形成细胞与细胞外基质替代物混合制成的人工皮肤。组织工程化皮肤除在临床使用以外,由于其稳定性高、操作简便也促进了其在非临床研究工作中的开展。在皮肤生物学研究领域,皮肤衰老、光保护、代替动物测试实验等领域,体外构建的三维皮肤均具有很高的应用价值。三维皮肤模型可以满足不同细胞间的相互作用,可以为in vitro与in vivo之间提供一个重要的桥梁。
本发明方法可以有效建立三维皮肤模型,而且制备方法简便,可以用于皮肤抗炎抗敏药物的筛选,应用前景优良。
根据本发明的上述内容,按照本发明相关领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
附图说明
图1A、C生长不良成纤维细胞;B、D生长良好的成纤维细胞
图2A、C生长不良角质形成细胞;B、D生长良好的角质形成细胞
图3A单独HyStemTM(巯基修饰的透明质酸HA);B单独ExtralinkTM(聚乙二醇二丙烯酸酯,PEGDA);C单独I型胶原;D本发明透明质酸-胶原蛋白水凝胶
图4三维皮肤模型及HE染色
图5三维皮肤模型模拟图
图6全皮层皮肤模型的构建示意图
图7外周血来源免疫细胞培养
图8含有原代外周血来源免疫细胞(单核细胞、淋巴细胞、粒细胞等)的三维皮肤模型培养
图9含有肥大细胞三维皮肤模型如
图10含肥大细胞三维皮肤模型
图11炎症因子检测指标(固相芯片)
图12HE染色、扫描电镜观察
图13经受试物处理后细胞存活率
具体实施方式
实施例1本发明三维皮肤模型的制备
在体外将皮肤全层包括免疫细胞(淋巴细胞、树突状细胞、粒细胞、单核细胞、巨噬细胞等)的所有类型细胞以及生物支架材料复合构建出具有与正常皮肤结构形态相似皮肤的方法。
1本发明三维皮肤模型的构建方法
1.1各种细胞的来源和制备
1)成纤维细胞和角质形成细胞的获得:
取正常人的皮肤组织,将组织进行消毒,分离出真皮层组织与表皮层组织小块(3mm×3mm)。将组织小块放于无菌培养皿中,添加培养基(含有10%FBS的DMEM培养基)。组织小块贴壁于培养皿的贴面处,一部分细胞会贴在培养皿表面生长,其间每2天更换一次培养液,并通过显微镜观察其生长情况,通过6天的培养后,回收细胞,通过流式细胞仪对细胞进行筛选,获得成纤维细胞和角质形成细胞。前述方法种,细胞培养的条件为37℃、5%CO2培养箱中培养
获得成纤维细胞和角质形成细胞的生长图如图1B、D、图2B、D所示。
2)原代外周血细胞的获得:
无菌采集静脉血6ml注入肝素抗凝管中,轻轻摇匀,室温下加入等体积的PBS或Hank,s液,轻轻吹打混匀。6ml淋巴细胞分层液置于离心管中,再将稀释后的血样品在分离液面上方,18-20℃,2000rpm,离心30min,离心后从管底至液面分四层,依次为红细胞和粒细胞层、分层液层、单个核细胞层、血浆层。
其中单个核细胞培养方法为:将上层淡黄色的稀释血浆移除,轻轻吸出乳白色的单个核细胞层,放入新的离心管中,加入PBS洗涤两次,加RPMI-1640培养基(含10%FBS)培养单个核细胞。
其中单核细胞培养方法为:将上述单个核细胞移入培养瓶中培养(RPMI-1640培养基),贴壁2h后,吸去未贴壁的细胞,洗下未贴牢的细胞,刮下贴壁细胞为单核细胞,加RPMI-1640培养基(含10%FBS)培养单核细胞。
其中粒细胞培养方法为:依次将上层淡黄色的稀释血浆移除,乳白色的单个核细胞层移除,分离层移除,轻轻吸取出粒细胞,放入新的离心管中,加入PBS洗涤两次,加RPMI-1640培养基(含10%FBS)培养粒细胞。
其中树突状细胞培养方法为:上述单个核细胞移入培养瓶(RPMI-1640培养基)中培养,贴壁2h后,吸去未贴壁的细胞,收集贴壁的单核细胞,免疫磁珠法分离CD14+单核细胞,用含有10%FBS、50ng/mL rhGM-CSF和25ng/mL IL-4的RPMI-1640培养液置于37℃、5%CO2培养箱中培养,5-6d后细胞即可分化为树突状细胞。
其中T淋巴细胞分离方法为:上述单个核细胞移入培养瓶中培养,贴壁2h后,收集未贴壁的细胞,加入含10%FBS的RPMI-1640培养液,即得T淋巴细胞。
其中巨噬细胞的培养方法为:上述单核细胞移入培养瓶中培养1-2d,加入终浓度为125ng/mL的佛波酯(PMA)的RPMI-1640培养基进行细胞分化,培养2-3d,即分化为巨噬细胞。
外周血来源免疫细胞培养如图7所示。
3)原代肥大细胞的获得
手术切取的人瘢痕组织,除去皮下脂肪,用Tyrode液清晰皮片并切成1mm2大小的小块,加入消化液(0.3mg/ml的Ⅰ型胶原酶和100U/ml的透明质酸酶)消化4h,再剪切至乳白色悬液,过滤收集滤液。在4℃条件下离心滤液,弃上清液,用冷的Tyrode液重新悬浮沉淀,再离心,弃上清液,将悬浮细胞接种入含10%FBS的DMEM培养液,即得肥大细胞。
1.2建模方法
1)制备细胞外基质高分子水凝胶溶液:
透明质酸-胶原蛋白水凝胶:HyStem试剂盒(Sigma)内冻干状固体HyStemTM(巯基修饰的透明质酸HA)和ExtralinkTM(聚乙二醇二丙烯酸酯,PEGDA)分别用去离子水溶解后获得10mg/mL储存液,I型胶原(Sigma)配制为10mg/ml,pH值为7.2的存储液。HA、PEGDA与I型胶原(4:1:1)混合均匀后,即得水凝胶溶液。
水凝胶溶液凝固,即可得到水凝胶三维支架,其示意图如图3D所示。
2)构建三维皮肤模型
a、第一种:全皮层皮肤模型
构建示意图如图6所示:
将成纤维细胞接种入水凝胶溶液中,每1ml水凝胶溶液加入5×105~8×105个细胞,轻轻放入培养小室(transwell)中移入12孔培养板中,置于培养箱进行培养,30分钟后水凝胶溶液固化形成水凝胶,即为含有成纤维细胞的支架,添加DMEM培养基淹没培养小室,培养构建真皮,培养5日后,在其支架表面接种角质形成细胞(每孔加入2×105~4×105),通过气-液界面培养形成表皮:具体是先在DMEM培养基淹没培养小室的情况下培养5日,然后在在DMEM培养基处于培养小室底部的情况下培养14日,即制备得到。
对皮肤模型进行石蜡包埋处理后,进行HE染色,与正常人皮肤进行形态学对比,判断其培养状况。三维皮肤模型及HE染色如图4所示;三维皮肤模型模拟图如图5所示。
b、第二种:含外周血来源免疫细胞的三维皮肤模型
构建示意图如图8所示:
将成纤维细胞接种入水凝胶溶液中,每1ml水凝胶溶液加入5×105~8×105个细胞,轻轻放入培养小室(transwell)中移入12孔培养板中,置于培养箱进行培养,30分钟后水凝胶溶液固化形成水凝胶,即为含有成纤维细胞的支架,添加DMEM培养基淹没培养小室,培养构建真皮,培养5日后,在其支架表面接种角质形成细胞(每孔加入2×105~4×105个细胞),通过气-液界面培养形成表皮:具体是先在DMEM培养基淹没培养小室的情况下培养1日,加入免疫细胞(单核细胞、树突状细胞、T淋巴细胞或者巨噬细胞)至培养小室的周围,每孔加入2×105~5×105个细胞继续培养4日,然后在DMEM培养基处于培养小室底部的情况下培养14日,即制备得到。
c、第三种:含有肥大细胞的三维皮肤模型
构建示意图如图9所示:
将成纤维细胞、肥大细胞接种入水凝胶溶液中,每1ml水凝胶溶液加入5×105~8×105个成纤维细胞和1.25×105~2×105个肥大细胞,轻轻放入培养小室(transwell)中移入12孔培养板中,置于培养箱进行培养,30分钟后水凝胶溶液固化形成水凝胶,即为含有成纤维细胞的支架,添加DMEM培养基淹没培养小室,培养构建真皮,培养5日后,在其支架表面接种角质形成细胞(每孔加入2×105~4×105),通过气-液界面培养形成表皮:具体是先在DMEM培养基淹没培养小室的情况下培养5日,然后在DMEM培养基处于培养小室底部的情况下培养14日,即制备得到。
2模型检测
1)HE染色和检测方法所构建组织工程皮肤观察所构建的皮肤结构以及各种免疫细胞的形成情况;
a)含有原代外周血来源免疫细胞(单核细胞、淋巴细胞、粒细胞等)的三维皮肤模型
培养24-72h将皮肤模型与免疫细细胞分离,并用生理盐水将皮肤模型表面冲洗干净并准备进行HE染色;分离后的免疫细胞用于检测细胞表面标志物的表达;培养上清用于炎症因子检测。
HE染色:取中性甲醛固定之标本常规石蜡包埋,在石蜡切片机上做连续切片,裱于经APES处理的玻片上,切片置于60℃恒温烤箱内烘烤,HE染色后封固,光镜观察。
流式细胞术检测外周血来源免疫细胞(单核细胞、淋巴细胞、粒细胞等)的参数:从细胞培养板中取出细胞,每孔分别移入相应EP管中,使用离心机离心(250g,5min,4℃)收集细胞,用FACS缓冲液(PBS+0.1%的BSA)清洗一次,重悬细胞。再加入含有单克隆抗体稀释液,室温避光反应20m in,离心去上清液,加PBS液稀释混匀后上机检测,采用FACS-Calibur(美国BD公司产品),氩离子激光,功率为15mw,激发光波长488nm。
检测结果:
中性粒细胞:Gr-1,cD11b
成熟T细胞(CD3+)、T辅助诱导细胞(CD3+/CD4+)、
单核细胞CD 14+
粒细胞CD 15+
树突状细胞:CD11c+,MHCII+
巨噬细胞:CD 14+,CD11b+
b)含有肥大细胞的三维皮肤模型
HE染色:取中性甲醛固定之标本常规石蜡包埋,在石蜡切片机上做连续切片,裱于经APES处理的玻片上,切片置于60℃恒温烤箱内烘烤,HE染色后封固,光镜观察。
肥大细胞(甲苯胺蓝)染色:石蜡切片常规脱蜡至水,甲苯胺蓝染液中30min后稍水洗,冰醋酸液中分化,蒸馏水洗后吹干,二甲苯透明,中性树胶封固后光镜下观察。
肥大细胞三维皮肤模型如图10。
2)用已知刺激物检测模型,检测生物标记物表达的改变。
刺激物对照组:脂多糖(LPS)、2,4-壬二烯
保护物对照组:米诺地尔
阴性对照物:双蒸水
将预孵育的皮肤模型(第二种:含外周血来源免疫细胞的三维皮肤模型)置于接种置于12孔细胞培养板中;将样品受试物涂抹于皮肤模型表面,于培养箱中后孵育24h,每组样品进行3组平行试验;24h后用生理盐水将皮肤模型表面受试物冲洗干净并准备进行MTT测试,扫描电镜观察,HE染色,流式细胞检测相关指标变化;上清液采用固相芯片ProteomeProfiler Human Cytokine Array Kit(BD)检测相关炎症因子的表达。炎症因子检测指标如图11所示。
结果如图12~13所示:
通过监测暴露受试物后的细胞活率、形态变化、细胞因子表达情况、细胞表面标志物表达情况和相对荧光强度,判断化学物或个人护理产品的皮肤抗炎抗敏功效。
对三维皮肤模型上添加500uM的2,4-壬二烯醛或2ug/ml LPS培养6小时,然后除去培养液,重新添加培养液48h,之后制作成切片进行观察拍摄。对比无处理对照组,在纵剖切片上观察到较多由于***反应和炎症引起的皮肤空洞;在横剖切片上观察到皮肤表面形成了较多的鳞屑状皮损。LPS和2,4-壬二烯醛诱发的三维皮肤的损伤了皮肤的整体,对受损三维皮肤模型添加0.1mM的米诺地尔,对上侧培养6小时,皮肤空洞与表面鳞屑皮损得到了有效的修复。
采用前述方法对前述第一种全皮层皮肤模型和第三种含有肥大细胞的三维皮肤模型进行检测,LPS和2,4-壬二烯醛同样可以诱发三维皮肤的损伤,对受损三维皮肤模型添加0.1mM的米诺地尔,对上侧培养6小时,皮肤空洞与表面鳞屑皮损也得到了有效的修复。
实验结果说明,本发明三维皮肤模型建立成功,可用于皮肤抗炎抗敏药物的筛选。
该实验证明,本发明建立了三维皮肤模型,可用于筛选皮肤抗炎抗敏药物,应用前景优良。
Claims (9)
1.一种用于筛选治疗皮肤抗炎抗敏药物的三维皮肤模型的建立方法,其特征在于:包括如下步骤:
1)制备细胞外基质高分子水凝胶溶液;所述细胞外基质高分子水凝胶溶液是透明质酸、聚乙二醇二丙烯酸酯和I型胶原的混合溶液,其中,透明质酸、聚乙二醇二丙烯酸酯和I型胶原的重量比为4:1:1,透明质酸、聚乙二醇二丙烯酸酯和I型胶原的浓度分别为6.67mg/ml、1.67 mg/ml、1.67 mg/ml;
2)将成纤维细胞与细胞外基质高分子水凝胶溶液混合,放入培养小室中培养;
3)接种角质形成细胞,通过气-液界面培养,得到包含真皮层、表皮层和角质层的三维皮肤模型;气-液界面培养的方法是:接种角质形成细胞后,先在DMEM培养基淹没培养小室的情况下培养1日,加入免疫细胞至培养小室的周围,继续培养4日,然后在DMEM培养基处于培养小室底部的情况下培养14日,即制备得到;接种的免疫细胞的量为:步骤2)中采用12孔板培养时,每孔加入2×105~5×105免疫细胞,所述免疫细胞为单核细胞、树突状细胞、T 淋巴细胞或者巨噬细胞。
2.根据权利要求1所述的方法,其特征在于:步骤2)中,将成纤维细胞与细胞外基质高分子水凝胶溶液混合的同时,加入1.25×105~2×105个肥大细胞,混合。
3.根据权利要求2所述的方法,其特征在于:所述肥大细胞按照如下方法制备:取瘢痕组织,除去皮下脂肪,切成小块,加入消化液消化4 h,再剪切至乳白色悬液,过滤收集滤液;在4℃条件下离心滤液,弃上清液,重悬,再离心,弃上清液,将悬浮细胞接种入含10% FBS的DMEM培养液中培养,即得肥大细胞;所述消化液是Ⅰ型胶原酶和透明质酸酶的混合溶液,其中Ⅰ型胶原酶的浓度为0.3mg/ml,透明质酸酶的浓度为100U/ml。
4.根据权利要求1~3任一项所述的方法,其特征在于:步骤2)中,每1ml水凝胶溶液加入5×105~8×105个成纤维细胞,放入培养小室中,置于培养板中培养,培养30min后形成水凝胶,培养的时间为5日。
5.根据权利要求1所述的方法,其特征在于:步骤3)中,接种的角质形成细胞的量为:步骤2)中采用12孔板培养时,每孔加入2×105~4×105角质形成细胞。
6.根据权利要求1所述的方法,其特征在于:所述成纤维细胞按照如下方法制备:取皮肤真皮层组织,切成小块,采用含有10%FBS的DMEM培养基培养,其间每2天更换一次培养液,培养 6天后,回收细胞,通过流式细胞仪对细胞筛选成纤维细胞,即可;
所述角质形成细胞按照如下方法制备:取皮肤表皮层组织,切成小块,采用含有10%FBS的DMEM培养基培养,其间每2天更换一次培养液,培养 6天后,回收细胞,通过流式细胞仪对细胞筛选角质形成细胞,即可;
所述单核细胞按照如下方法制备:取血液,采用淋巴细胞分层液处理,离心,取单个核细胞层,采用RPMI-1640培养基,贴壁 2h 后,去除未贴壁或者未贴牢的细胞,刮下的贴壁细胞为单核细胞;
所述粒细胞按照如下方法制备:取血液,采用淋巴细胞分层液处理,离心,取粒细胞层,洗涤,采用RPMI-1640培养基培养,即得;
所述树突状细胞按照如下方法制备:取血液,采用淋巴细胞分层液处理,离心,取单个核细胞层,采用RPMI-1640培养基,贴壁 2h 后,去除未贴壁或者未贴牢的细胞,收集贴壁细胞,免疫磁珠法分离 CD14+ 单核细胞, 用含有10% FBS、 50ng/mL rhGM-CSF 和 25ng/mLIL-4 的 RPMI-1640培养液置于 37℃、 5% CO2培养箱中培养 5-6d ,得树突状细胞;
所述T 淋巴细胞按照如下方法制备:取血液,采用淋巴细胞分层液处理,离心,取单个核细胞层,采用RPMI-1640培养基,贴壁 2h 后,收集未贴壁的细胞,加入含 10% FBS的RPMI-1640 培养液,即得T 淋巴细胞;
所述巨噬细胞按照如下方法制备:取血液,采用淋巴细胞分层液处理,离心,取单个核细胞层,采用RPMI-1640培养基培养1-2d,加入终浓度为 125 ng/mL 的佛波酯(PMA)的RPMI-1640培养基进行细胞分化,培养 2-3d,即得巨噬细胞。
7.权利要求1~6任意一项所述方法建立的三维皮肤模型。
8.权利要求7所述三维皮肤模型在筛选皮肤抗炎抗敏药物中的用途。
9.一种筛选治疗皮肤抗炎抗敏药物的方法,其特征在于:包括如下步骤:
a、按照权利要求1~6任意一项所述的方法建立的三维皮肤模型;
b、将候选物施用于a步骤所述的三维皮肤模型;
c、用三维皮肤模型评价潜在的抗炎抗敏药物。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710458682.4A CN107267441B (zh) | 2017-06-16 | 2017-06-16 | 一种建立用于抗炎抗敏功效评价的三维皮肤模型的方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710458682.4A CN107267441B (zh) | 2017-06-16 | 2017-06-16 | 一种建立用于抗炎抗敏功效评价的三维皮肤模型的方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN107267441A CN107267441A (zh) | 2017-10-20 |
CN107267441B true CN107267441B (zh) | 2021-04-23 |
Family
ID=60067259
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201710458682.4A Active CN107267441B (zh) | 2017-06-16 | 2017-06-16 | 一种建立用于抗炎抗敏功效评价的三维皮肤模型的方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN107267441B (zh) |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110940813A (zh) * | 2019-10-31 | 2020-03-31 | 广州市华代生物科技有限公司 | 一种采用重建正常人体三维皮肤模型评价修复功能的体外方法 |
CN111073844A (zh) * | 2019-11-29 | 2020-04-28 | 广州市华代生物科技有限公司 | 一种皮肤单器官芯片长期培养模型的制备方法 |
CN113699202A (zh) * | 2020-05-21 | 2021-11-26 | 华子昂 | 一种用混合细胞与人工细胞培养巢制备胶原蛋白的方法 |
CN113564114B (zh) * | 2021-01-16 | 2023-04-07 | 浙江工商大学 | 一种cmt93-dc-t细胞的三维细胞模型的构建方法和应用 |
CN112961899B (zh) * | 2021-02-23 | 2023-06-09 | 云南贝泰妮生物科技集团股份有限公司 | 一种化妆品原料体外巨噬细胞联合3d皮肤模型抗炎功效筛选方法 |
CN113549670A (zh) * | 2021-07-20 | 2021-10-26 | 云南贝泰妮生物科技集团股份有限公司 | 一种体外细胞与3d表皮模型共培养的化妆品致敏评价方法 |
CN113652392B (zh) * | 2021-08-17 | 2023-09-26 | 云南贝泰妮生物科技集团股份有限公司 | 一种基于体外巨噬细胞与3d皮肤模型共培养的化妆品抗炎功效评估方法 |
CN116162585A (zh) * | 2021-11-24 | 2023-05-26 | 青岛蔚蓝生物集团有限公司 | 一种用HaCaT细胞构建的皮肤细胞紫外线损伤模型 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6497875B1 (en) * | 1996-04-26 | 2002-12-24 | Case Western Reserve University | Multilayer skin or dermal equivalent having a layer containing mesenchymal stem cells |
CN101959908A (zh) * | 2008-03-04 | 2011-01-26 | Jsr株式会社 | 放射线敏感性组合物和聚合物以及单体 |
KR101103571B1 (ko) * | 2009-09-23 | 2012-01-09 | 한림대학교 산학협력단 | 피부조직 재생용 구조체 및 이를 이용한 인공피부 |
EP3167913A1 (en) * | 2014-07-09 | 2017-05-17 | Jeon, Saewha | Method for making three-dimensional cultured skin model including dermis and epidermis, and three-dimensional cultured skin model made thereby |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP6033023B2 (ja) * | 2012-09-25 | 2016-11-30 | 株式会社ジェイメック | 色素含有人工皮膚 |
-
2017
- 2017-06-16 CN CN201710458682.4A patent/CN107267441B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6497875B1 (en) * | 1996-04-26 | 2002-12-24 | Case Western Reserve University | Multilayer skin or dermal equivalent having a layer containing mesenchymal stem cells |
CN101959908A (zh) * | 2008-03-04 | 2011-01-26 | Jsr株式会社 | 放射线敏感性组合物和聚合物以及单体 |
KR101103571B1 (ko) * | 2009-09-23 | 2012-01-09 | 한림대학교 산학협력단 | 피부조직 재생용 구조체 및 이를 이용한 인공피부 |
EP3167913A1 (en) * | 2014-07-09 | 2017-05-17 | Jeon, Saewha | Method for making three-dimensional cultured skin model including dermis and epidermis, and three-dimensional cultured skin model made thereby |
Non-Patent Citations (4)
Title |
---|
化学品刺激性组织工程皮肤检测模型的构建;张兆清等;《中国工业医学杂志》;20060430;第19卷(第2期);81-83 * |
张兆清等.化学品刺激性组织工程皮肤检测模型的构建.《中国工业医学杂志》.2006,第19卷(第2期),81-83. * |
透明质酸/胶原蛋白/聚乙二醇复合水凝胶的研究;袁静等;《功能材料》;20151231;第46卷(第15期);15052-15057 * |
重组人皮肤模型评价化妆品皮肤腐蚀性/刺激性替代方法的建立;刘诊等;《卫生研究》;20090731;第38卷(第4期);468-471 * |
Also Published As
Publication number | Publication date |
---|---|
CN107267441A (zh) | 2017-10-20 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107267441B (zh) | 一种建立用于抗炎抗敏功效评价的三维皮肤模型的方法 | |
Yoon et al. | Development of cell-laden 3D scaffolds for efficient engineered skin substitutes by collagen gelation | |
JP6712016B2 (ja) | 三次元組織体及びその製造方法、並びに、三次元組織体の形成剤 | |
CN109432127A (zh) | 间充质干细胞外泌体在制备促毛发再生的药物制剂中的应用 | |
DK2471902T3 (en) | Preparation of artificial tissues by tissue engineering with agarose-fibrin biomaterials | |
KR20100065338A (ko) | 인간 또는 동물 배아에서 중간엽 줄기세포를 추출 및 그 분비물을 추출하는 방법 | |
CN111826340B (zh) | 一种抗皮肤衰老的鹿茸干细胞外泌体的制备方法及其应用 | |
CN108753708B (zh) | 一种干细胞活性因子冻干粉的制备方法 | |
JP2009167206A (ja) | invitroにおけるCD14陽性単球からの樹状細胞の産出 | |
KR102159915B1 (ko) | 엑소좀 및/또는 세포외 소포체의 생성 촉진 방법 | |
CN106801038A (zh) | 一种利用三维细胞培养***促进脐带血造血干细胞快速稳定增殖的细胞培养方法 | |
CN106244533A (zh) | 牙龈间充质干细胞的原代分离方法 | |
CN110693912A (zh) | 干细胞外泌体在制备促进创面愈合的产品中的应用 | |
KR20040076860A (ko) | 인간 단핵 식세포성 백혈구의 제조 방법 | |
Pajoum et al. | In vitro co-culture of human skin keratinocytes and fibroblasts on a biocompatible and biodegradable scaffold | |
Gunawan et al. | Isolation of human skin dendritic cell subsets | |
CN105087466B (zh) | 诱导脐带间充质干细胞向角膜上皮细胞分化的培养基和方法 | |
JP2010502175A (ja) | Cd14+単球からランゲルハンス細胞及び/又は真皮/間質性樹状細胞を製造する方法 | |
GENG et al. | Lipofuscin accumulation in iris pigment epithelial cells exposed to photoreceptor outer segments | |
Gea et al. | Study of bacterial cellulose as scaffold on cartilage tissue engineering | |
CN105087482B (zh) | 一种细胞培养基质及其应用与使用方法 | |
KR102161946B1 (ko) | 측분비인자 및 이의 제조방법 | |
KR102161947B1 (ko) | 측분비인자를 포함하는 세포 배양용 조성물 | |
CN113018501A (zh) | 一种内皮祖细胞外泌体医用敷料、制备方法及其应用 | |
DK2585585T3 (en) | Method for harvesting cells grown in 3D hydrogel matrices |
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 | ||
GR01 | Patent grant | ||
GR01 | Patent grant |