CN113293126B - 一种人***粘膜模型的体外构建方法 - Google Patents
一种人***粘膜模型的体外构建方法 Download PDFInfo
- Publication number
- CN113293126B CN113293126B CN202110356820.4A CN202110356820A CN113293126B CN 113293126 B CN113293126 B CN 113293126B CN 202110356820 A CN202110356820 A CN 202110356820A CN 113293126 B CN113293126 B CN 113293126B
- Authority
- CN
- China
- Prior art keywords
- vaginal
- culture
- solution
- cells
- hve
- 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
- 210000004877 mucosa Anatomy 0.000 title claims abstract description 61
- 238000010276 construction Methods 0.000 title claims abstract description 26
- 238000000338 in vitro Methods 0.000 title claims abstract description 20
- 210000004027 cell Anatomy 0.000 claims abstract description 66
- 210000002919 epithelial cell Anatomy 0.000 claims abstract description 51
- 102000008186 Collagen Human genes 0.000 claims abstract description 38
- 108010035532 Collagen Proteins 0.000 claims abstract description 38
- 229920001436 collagen Polymers 0.000 claims abstract description 38
- 210000002950 fibroblast Anatomy 0.000 claims abstract description 34
- 239000011159 matrix material Substances 0.000 claims abstract description 29
- 210000001519 tissue Anatomy 0.000 claims abstract description 23
- 239000000243 solution Substances 0.000 claims description 121
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 46
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 claims description 40
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims description 40
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 38
- 238000012258 culturing Methods 0.000 claims description 36
- 239000012091 fetal bovine serum Substances 0.000 claims description 32
- 238000000034 method Methods 0.000 claims description 30
- 239000001963 growth medium Substances 0.000 claims description 26
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 25
- 238000004113 cell culture Methods 0.000 claims description 22
- 239000006285 cell suspension Substances 0.000 claims description 21
- 210000005081 epithelial layer Anatomy 0.000 claims description 21
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 20
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 20
- 102000004877 Insulin Human genes 0.000 claims description 20
- 108090001061 Insulin Proteins 0.000 claims description 20
- 229960000890 hydrocortisone Drugs 0.000 claims description 20
- 229940125396 insulin Drugs 0.000 claims description 20
- 208000025337 Vulvar squamous cell carcinoma Diseases 0.000 claims description 18
- 230000029087 digestion Effects 0.000 claims description 18
- 239000002585 base Substances 0.000 claims description 17
- 229930024421 Adenine Natural products 0.000 claims description 15
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 15
- 102000018233 Fibroblast Growth Factor Human genes 0.000 claims description 15
- 108050007372 Fibroblast Growth Factor Proteins 0.000 claims description 15
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 102000004338 Transferrin Human genes 0.000 claims description 15
- 108090000901 Transferrin Proteins 0.000 claims description 15
- 229960000643 adenine Drugs 0.000 claims description 15
- 229940126864 fibroblast growth factor Drugs 0.000 claims description 15
- 239000012581 transferrin Substances 0.000 claims description 15
- 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 claims description 15
- 102000009024 Epidermal Growth Factor Human genes 0.000 claims description 14
- 101800003838 Epidermal growth factor Proteins 0.000 claims description 14
- 229940116977 epidermal growth factor Drugs 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 11
- JWZZKOKVBUJMES-UHFFFAOYSA-N (+-)-Isoprenaline Chemical compound CC(C)NCC(O)C1=CC=C(O)C(O)=C1 JWZZKOKVBUJMES-UHFFFAOYSA-N 0.000 claims description 10
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 claims description 10
- AUYYCJSJGJYCDS-LBPRGKRZSA-N Thyrolar Chemical compound IC1=CC(C[C@H](N)C(O)=O)=CC(I)=C1OC1=CC=C(O)C(I)=C1 AUYYCJSJGJYCDS-LBPRGKRZSA-N 0.000 claims description 10
- 229930003268 Vitamin C Natural products 0.000 claims description 10
- 229960001317 isoprenaline Drugs 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- 210000004400 mucous membrane Anatomy 0.000 claims description 10
- 230000007935 neutral effect Effects 0.000 claims description 10
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 claims description 10
- 229940035722 triiodothyronine Drugs 0.000 claims description 10
- 235000019154 vitamin C Nutrition 0.000 claims description 10
- 239000011718 vitamin C Substances 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 9
- 239000002609 medium Substances 0.000 claims description 7
- 230000001376 precipitating effect Effects 0.000 claims description 7
- 210000002966 serum Anatomy 0.000 claims description 7
- 239000012894 fetal calf serum Substances 0.000 claims description 6
- 102000029816 Collagenase Human genes 0.000 claims description 5
- 108060005980 Collagenase Proteins 0.000 claims description 5
- 239000003637 basic solution Substances 0.000 claims description 5
- 244000309466 calf Species 0.000 claims description 5
- 229960002424 collagenase Drugs 0.000 claims description 5
- 238000004090 dissolution Methods 0.000 claims description 5
- 238000011081 inoculation Methods 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 229940054269 sodium pyruvate Drugs 0.000 claims description 5
- 238000005303 weighing Methods 0.000 claims description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 4
- 238000005520 cutting process Methods 0.000 claims description 4
- 229940088598 enzyme Drugs 0.000 claims 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 claims description 4
- 229930182555 Penicillin Natural products 0.000 claims 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 claims description 2
- 230000001079 digestive effect Effects 0.000 claims description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 2
- 229940049954 penicillin Drugs 0.000 claims description 2
- 206010041823 squamous cell carcinoma Diseases 0.000 claims description 2
- 229960005322 streptomycin Drugs 0.000 claims description 2
- 206010047786 Vulvovaginal discomfort Diseases 0.000 abstract description 4
- 238000002474 experimental method Methods 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 238000001727 in vivo Methods 0.000 abstract description 2
- 235000016709 nutrition Nutrition 0.000 abstract description 2
- 230000015572 biosynthetic process Effects 0.000 abstract 1
- 238000012136 culture method Methods 0.000 abstract 1
- 230000035764 nutrition Effects 0.000 abstract 1
- 239000010410 layer Substances 0.000 description 50
- 210000000981 epithelium Anatomy 0.000 description 15
- 201000008190 vulva squamous cell carcinoma Diseases 0.000 description 10
- 238000002955 isolation Methods 0.000 description 7
- 229960000583 acetic acid Drugs 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 5
- 239000000758 substrate Substances 0.000 description 4
- 230000004888 barrier function Effects 0.000 description 3
- 239000002537 cosmetic Substances 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001817 pituitary effect Effects 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 210000005000 reproductive tract Anatomy 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 210000000434 stratum corneum Anatomy 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 101100394003 Butyrivibrio fibrisolvens end1 gene Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 1
- -1 E6E7 Proteins 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101100334732 Mus musculus Fgfr2 gene Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 231100000950 SkinEthic RHE Toxicity 0.000 description 1
- 206010070863 Toxicity to various agents Diseases 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229960002713 calcium chloride Drugs 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000012930 cell culture fluid Substances 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000003433 contraceptive agent Substances 0.000 description 1
- 230000002254 contraceptive effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000004177 elastic tissue Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 210000001821 langerhans cell Anatomy 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000003641 microbiacidal effect Effects 0.000 description 1
- 230000009525 mild injury Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000002035 prolonged 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
- 230000001105 regulatory effect Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000013334 tissue model Methods 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 210000003905 vulva Anatomy 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
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle 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
- C12N5/0693—Tumour cells; Cancer 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
- 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
- C12N2500/14—Calcium; Ca chelators; Calcitonin
-
- 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
- C12N2500/20—Transition metals
- C12N2500/24—Iron; Fe chelators; Transferrin
-
- 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
- C12N2500/40—Nucleotides, nucleosides, bases
-
- 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
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
-
- 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
-
- 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
- C12N2501/30—Hormones
-
- 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
- C12N2501/30—Hormones
- C12N2501/33—Insulin
-
- 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
-
- 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)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Reproductive Health (AREA)
- Oncology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供了一种人***粘膜模型的体外构建方法,采用***成纤维细胞和胶原构建基质层,在此基础上接种***上皮细胞,形成具有双层结构的体外***粘膜模型,在结构上与天然组织高度相似,在该模型上进行产品的***刺激性实验更加接近于体内真实情况,实验结果更准确可靠。***上皮细胞和基质层共培养时采用液下培养和气液面培养两个阶段,并且在不同的培养阶段针对细胞增殖和分化的不同需求对培养液的营养成分进行了精细化的调节,这种分段式培养法,既能保证细胞在不同发育阶段的营养需求,有利于复层化***粘膜上皮结构的形成,又能缩短构建时间、降低生产成本。
Description
技术领域
本发明属于生物医学领域,尤其涉及一种人***粘膜模型的体外构建方法。
背景技术
人的***粘膜由厚的,非角化的,分层的鳞状上皮组成,富含糖原,以及少量其他类型的细胞,例如巨噬细胞和朗格汉斯细胞。上皮细胞下面为固有层,包含许多弹性纤维和密集的血管网络,因此***粘膜是为局部和全身治疗输送药物的绝佳途径。但是由于长期暴露于生殖道管腔内的各种病原体中,***上皮也是许多病原体进入体内的潜在部位,例如在使用女性护理和美容产品,避孕药具或杀微生物剂之后,可能会造成轻度伤害,这可能会引起组织刺激,并使***上皮特别容易受到各种类型的感染。药品,化妆品和个人护理产品等专门配制用于人***粘膜的配方有时也会引起不良的局部或全身性副作用。因此,在产品投放市场之前评估新开发的化妆品、个人护理产品或局部应用药物与人***粘膜表面的相容性是制造商需要解决的关键问题。
分层分化的人类***上皮模型具有克服细胞单层膜的某些缺点的潜力,因为前者包含屏障层并允许局部施用活性成分和最终制剂,包括那些非水溶性的成分。除了可以避免动物福利风险和物种差异问题之外,体外组织模型通常还可以区分动物模型对其不敏感的非常温和的产品。
法国SkinEthic公司的RHVE模型采用外阴表皮样癌细胞系A431为种子细胞,细胞系稳定可靠,可以解决种子细胞来源困难问题,但只包含由A431细胞组成的上皮层,缺少基质层细胞和基质,因此在组织结构上还是与天然***组织存在较大差异。美国MatTek公司的EpiVaginaL***模型和中国专利CN201710048227.7中公开的人***上皮3D模型组织结构与天然***粘膜高度相似,然而由于可用的***组织来源有限,而且***黏膜上皮为复层鳞状上皮,大部分细胞属于分化成熟细胞,增殖能力较低,仅少部分基底层细胞具有较强的增殖能力,因此体外大量扩增培养条件要求较高,无法实现体外大量生产,满足市场需求。中国专利CN201710048227.7中公开的人***上皮3D模型还存在一些缺陷,如培养体系单一不能适应细胞不同阶段的营养需求,导致模型复层化较差,屏障功能较弱,而且该模型构建时间长,体外构建一批模型需要长达一个月时间,过长的生产周期会增加过程中的不可控因素,影响模型的稳定性,目前该模型仅限于实验室培养,未进行产业化生产,所以国内目前尚未有商业化的人***粘膜模型。
发明内容
针对现有技术存在的问题,本发明提供了一种人***粘膜模型的体外构建方法。
为实现上述发明目的,本发明采用下述技术方案予以实现:
一种人***粘膜模型的体外构建方法,包括如下步骤:
A、含成纤维细胞的基质层构建:
(1)取第5~10代***成纤维细胞,用含20%胎牛血清的DMEM配成细胞悬液;
(2)将细胞悬液和中性胶原支架按照1:9的体积比例混匀,接种到细胞培养小室中首次培养,然后在小室内外分别添加HVE-1培养液,二次培养后形成***粘膜模型中的基质层;
所述HVE-1培养液按照以下方法配制而成:以DMEM和F12按体积比1~3:1混合的培养基为基础液,在其中添加10%胎牛血清,添加2.0~6.0μmol/mL的谷氨酰胺,15~30μg/mL腺嘌呤,5~15μg/mL胰岛素,0.5~2.0μg/mL 氢化可的松,25~100μg/mL维生素C,1~10ng/mL成纤维细胞生长因子和5~20ng/mL转铁蛋白;
B、细胞的接种:
吸去基质层表面的HVE-1培养液,将***上皮细胞或外阴表皮样癌细胞系A431细胞用HVE-2培养液配制成细胞悬液,接种到细胞培养小室中的基质层上,将培养基更换成HVE-2培养液进行浸没式培养;
所述HVE-2培养液按照以下方法配制而成:以DMEM和F12按体积比1~3:1混合的培养基为基础液,在其中添加5%胎牛血清,2.0~6.0μmol/mL的谷氨酰胺,15~30μg/mL腺嘌呤,5~15μg/mL胰岛素,0.5~2.0μg/mL 氢化可的松,1~10ng/mL成纤维细胞生长因子,5~20ng/mL转铁蛋白,0.5~2.0ng/mL 表皮生长因子,0.1~0.4nmol/mL三碘甲状腺原氨酸和5~10nmol/mL异丙肾上腺素;
C、***粘膜模型的培养:
吸去培养物表面的HVE-2培养液,将细胞培养小室提升至空气-液体界面进行气液面培养;将培养基更换成HVE-3培养液,培养后得到双层***粘膜模型。所述双层***粘膜模型为基质层中含有成纤维细胞,上皮层中含有***上皮细胞的双层***粘膜模型。
所述HVE-3培养液按照以下方法配制而成:以DMEM和F12按体积比1~3:1混合的培养基为基础液,在其中添加5%胎牛血清,2.0~6.0μmol/mL的谷氨酰胺,15~30μg/mL腺嘌呤,5~15μg/mL胰岛素,0.5~2.0μg/mL氢化可的松,1~10ng/mL成纤维细胞生长因子,5~20ng/mL转铁蛋白,2.0~5.0ng/mL 表皮生长因子,0.1~0.4nmol/mL三碘甲状腺原氨酸,5~10nmol/mL异丙肾上腺素,25~100μg/mL维生素C和1.0~1.5μmol/mL CaCl2。
优选地,步骤A中***成纤维细胞的分离和培养步骤如下:
(1)将分离的***组织固有层放入胶原酶消化液中消化,然后使用含10%胎牛血清的DMEM培养液终止消化,过滤后离心去上清,收集***成纤维细胞;
(2)PBS清洗***成纤维细胞,重悬细胞沉淀于含10%新生牛血清的DMEM培养液中,传代培养至第五代后用于进行***粘膜模型中基质层的构建。
优选地,步骤B中***上皮细胞的分离和培养步骤如下:
(1)将人体***组织置于培养皿中, PBS溶液清洗6次,除去粘膜下组织,将组织块切块加入分散酶中消化;
(2)眼科镊分离出***粘膜上皮层和固有层,将***粘膜上皮层放入胰酶-EDTA中消化;
(3)使用含10%胎牛血清的DMEM培养液终止消化,过滤后离心去上清,收集***粘膜上皮细胞;
(4)PBS清洗***粘膜上皮细胞,重悬细胞沉淀于无血清上皮细胞培养液中,获得分离成单个细胞的***上皮细胞悬液,传代培养至第二代得到纯度达95%以上的***上皮细胞。
优选地,步骤A中第(4)步所述无血清上皮细胞培养液为以DMEM和F12按体积比1~3:1混合的培养基为基础液,在其中添加2.0~6.0μmol/mL的谷氨酰胺,2.0~5.0ng/mL 表皮生长因子,15~30μg/mL 牛垂体提取物,10~15ng/mL胰岛素,0.5~2.0μg/mL 氢化可的松和0.1~0.4μmol/mL CaCl2。
本发明中的无血清上皮细胞培养液以DMEM/F12为基础液,包含谷氨酰胺、牛垂体提取物(BPE)、表皮生长因子(EGF)、胰岛素、氢化可的松、氯化钙等适宜浓度的因子和蛋白,适合用于培养***上皮细胞,包括从正常组织中提取的原代细胞以及各种永生化和转化的***上皮细胞系。本发明中的无血清上皮细胞培养液由于不含血清成份,可有效减少***上皮细胞提取过程中成纤维细胞的污染,传代培养至第二代***上皮细胞纯度达95%以上。此外,还可以适用于多种永生化和转化的***上皮细胞系的培养,有利于进行大规模生产,可以很好的节约人工和材料成本。
优选地,步骤B中外阴表皮样癌细胞系A431细胞的培养过程为:重悬外阴表皮样癌细胞系A431细胞沉淀于含10%胎牛血清、2.0~6.0μmol/mL谷氨酰胺、0.1~1μmol/mL丙酮酸钠和4.5g/L高糖的DMEM培养液,传代培养至第七代后用于进行上皮层的构建。
通过外阴表皮样癌细胞系A431细胞培养构建的双层***粘膜模型为基质层中含有成纤维细胞,上皮层中含有外阴表皮样癌细胞系A431的双层***粘膜模型。
优选地,步骤A中中性胶原支架的制备方法为:称量胶原置于0.1%醋酸溶液中配制成4~10mg/mL的胶原溶液,待完全溶解后置于冰上,将胶原溶液和含10%胎牛血清DEME培养液按体积比1:8混匀,加入0.1M的NaOH溶液调节PH至7.2~7.4。
优选地,步骤A中首次培养温度为37℃,首次培养时间为2~3小时,二次培养温度为37℃,二次培养时间为2~4天。
优选地,步骤B中培养温度为37℃,培养时间为4天。
优选地,步骤C中培养温度为37℃,培养时间为8~12天。
优选地,所述PBS溶液中含有100U/mL青霉素和100U/mL链霉素。
优选地,所述EDTA浓度为0.2mg/mL。
优选地,步骤C中采用去离子水将冰醋酸稀释为0.1%醋酸溶液,过滤除菌后使用。
优选地,本发明中用于***粘膜上皮层构建的种子细胞为正常原代***上皮细胞、永生化***上皮细胞系Ect1、E6E7、End1 、E6E7、Vk2 、E6E7或外阴表皮样癌细胞系A431中的一种或多种。种子细胞选择多样,体外构建重复性佳,解决种子细胞来源困难、细胞稳定性差等问题,可实现产业化制备。
相对于现有技术,本发明的有益效果在于:
本发明公开了一种人***粘膜模型的体外构建方法,采用***成纤维细胞和胶原构建基质层,在此基础上接种***上皮细胞,形成具有双层结构的体外***粘膜模型,在结构上与天然组织高度相似,在该模型上进行产品的***刺激性实验更加接近于体内真实情况,实验结果更准确可靠。本发明中***上皮细胞和基质层共培养时采用液下培养和气液面培养两个阶段,并且在不同的培养阶段针对细胞增殖和分化的不同需求对培养液的营养成分进行了精细化的调节,这种分段式培养法,既能保证细胞在不同发育阶段的营养需求,有利于复层化***粘膜上皮结构的形成,又能缩短构建时间、降低生产成本。
附图说明
图1A为本发明中使用正常人***上皮细胞培养构建的双层***粘膜上皮模型表观图片;
图1B为本发明中使用正常人***上皮细胞培养构建的双层***粘膜上皮模型组织学H&E染色图片;
图2A为本发明中使用外阴表皮样癌细胞系A431细胞培养构建的双层***粘膜上皮模型表观图片;
图2B为本发明中使用外阴表皮样癌细胞系A431细胞培养构建的双层***粘膜上皮模型组织学H&E染色图片。
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了,所述实施例仅仅是帮助理解本发明,不应视为对本发明的具体限制。
实施例1
一种人***粘膜模型的体外构建方法,包括如下步骤:
A、***上皮细胞的分离和培养:
(1)将人体***组织置于培养皿中,使用4℃预冷的PBS溶液清洗6次,除去粘膜下组织,将组织块切成0.2cm×0.2cm的大小,加入1.5U/mL的分散酶中,4℃消化过夜;
(2)眼科镊分离出***粘膜上皮层和固有层,将***粘膜上皮层放入0.25%胰酶-EDTA中,37℃消化30min,所述EDTA的浓度为0.2mg/mL;
(3)使用含10%胎牛血清的DMEM培养液终止消化,200目筛网过滤,离心去上清,收集***粘膜上皮细胞;
(4)PBS清洗***粘膜上皮细胞,重悬细胞沉淀于无血清上皮细胞培养液中,获得分离成单个细胞的***上皮细胞悬液,接种于T75培养瓶培养,37℃,5%CO2培养箱培养,传代培养至第二代得到纯度达95%以上的***上皮细胞;
上述无血清上皮细胞培养液按照以下方法配制而成:以DMEM和F12按体积比1:1混合的培养基为基础液,在其中添加2.0μmol/mL的谷氨酰胺,2.0ng/mL EGF,15μg/mL BPE,10ng/mL胰岛素,0.5μg/mL 氢化可的松,0.1μmol/mL CaCl2。
B、***成纤维细胞的分离和培养:
(1)将分离的***组织固有层放入200U/mL胶原酶消化液中,37℃消化3h,使用含10%胎牛血清的DMEM培养液终止消化,200目筛网过滤,离心去上清,收集***成纤维细胞;
(2)PBS清洗***成纤维细胞,重悬细胞沉淀于含10%新生牛血清的DMEM培养液中,接种于T75培养瓶培养,37℃,5%CO2条件下传代培养至第五代后用于进行***粘膜模型中基质层的构建;
C、中性胶原支架的制备:
称量胶原置于0.1%醋酸溶液中配制成4mg/mL的胶原溶液,待完全溶解后置于冰上,将胶原溶液和含10%胎牛血清DEME培养液按体积比1:8混匀,加入0.1M的NaOH溶液调节PH至7.2;
D、含成纤维细胞的基质层构建:
(1)取第5代***成纤维细胞,用含20%胎牛血清的DMEM配成密度为2×106个/mL细胞悬液;
(2)将细胞悬液和中性胶原支架按照1:9的体积比例混匀,接种到Transwell小室中,置于37℃条件下培养2小时,然后在Transwell小室内外分别添加HVE-1培养液,37℃,5%CO2培养箱培养2天,形成***粘膜模型中的基质层;
上述HVE-1培养液按照以下方法配制而成:以DMEM和F12按体积比1:1混合的培养基为基础液,在其中添加10%胎牛血清,2.0μmol/mL的谷氨酰胺,15μg/mL腺嘌呤,5μg/mL胰岛素,0.5μg/mL 氢化可的松,加入25μg/mL维生素C,1ng/mL成纤维细胞生长因子,5ng/mL转铁蛋白。
E、***上皮细胞的接种:
(1)吸去基质层表面的培养基,将上述得到的***上皮细胞或A431细胞系用HVE-2培养液配制成2.0×106个/mL细胞悬液,接种到Transwell小室中的基质层上;
(2)将培养基更换成HVE-2培养液进行浸没式培养,37℃、5%CO2培养箱中培养4天;
上述HVE-2培养液按照以下方法配制而成:以DMEM和F12按体积比1:1混合的培养基为基础液,在其中添加5%胎牛血清,2.0μmol/mL的谷氨酰胺,15μg/mL腺嘌呤,5μg/mL胰岛素,0.5μg/mL 氢化可的松,1ng/mL成纤维细胞生长因子,5ng/mL转铁蛋白,0.5ng/mLEGF,0.1nmol/mL三碘甲状腺原氨酸,5nmol/mL异丙肾上腺素。
F、***粘膜模型的培养:
(1)吸去Transwell小室中培养物表面的HVE-2培养液,将Transwell小室提升至空气-液体界面进行气液面培养;
(2)将HVE-2培养液更换成HVE-3培养液, 37℃、5%CO2培养箱中培养8天,得到基质层中含有成纤维细胞、上皮层中含有***上皮细胞的双层***粘膜模型。
上述HVE-3培养液按照以下方法配制而成:以DMEM和F12按体积比1:1混合的培养基为基础液,在其中添加5%胎牛血清,2.0μmol/mL的谷氨酰胺,15μg/mL腺嘌呤,5μg/mL胰岛素,0.5μg/mL 氢化可的松,1ng/mL成纤维细胞生长因子,5ng/mL转铁蛋白,2.0ng/mLEGF,0.1nmol/mL三碘甲状腺原氨酸,5nmol/mL异丙肾上腺素,25μg/mL维生素C,1.0μmol/mLCaCl2。
实施例2
一种人***粘膜模型的体外构建方法,包括如下步骤:
A、***上皮细胞的分离和培养:
(1)将人体***组织置于培养皿中,使用4℃预冷的PBS溶液清洗6次,除去粘膜下组织,将组织块切成0.2cm×0.2cm的大小,加入1.5U/mL的分散酶中,4℃消化过夜;
(2)眼科镊分离出***粘膜上皮层和固有层,将***粘膜上皮层放入0.25%胰酶-EDTA中,37℃消化45min,所述EDTA的浓度为0.2mg/mL;
(3)使用含10%胎牛血清的DMEM培养液终止消化,200目筛网过滤,离心去上清,收集***粘膜上皮细胞;
(4)PBS清洗***粘膜上皮细胞,重悬细胞沉淀于无血清上皮细胞培养液中,获得分离成单个细胞的***上皮细胞悬液,接种于T75培养瓶培养,37℃,5%CO2培养箱培养,传代培养至第二代得到纯度达95%以上的***上皮细胞;
上述无血清上皮细胞培养液按照以下方法配制而成:以DMEM和F12按体积比2:1混合的培养基为基础液,在其中添加4.0μmol/mL的谷氨酰胺,3.0ng/mL EGF,20μg/mL BPE,13ng/mL胰岛素,1.5μg/mL 氢化可的松,0.2μmol/mL CaCl2;
B、***成纤维细胞的分离和培养:
(1)将分离的***组织固有层放入200U/mL胶原酶消化液中,37℃消化3h,使用含10%胎牛血清的DMEM培养液终止消化,200目筛网过滤,离心去上清,收集***成纤维细胞;
(2)PBS清洗***成纤维细胞,重悬细胞沉淀于含10%新生牛血清的DMEM培养液中,接种于T75培养瓶培养,37℃,5%CO2条件下传代培养至第五代后用于进行***粘膜模型中基质层的构建;
C、中性胶原支架的制备:
称量胶原置于0.1%醋酸溶液中配制成6mg/mL的胶原溶液,待完全溶解后置于冰上,将胶原溶液和含10%胎牛血清DEME培养液按体积比1:8混匀,加入0.1M的NaOH溶液调节PH至7.3;
D、含成纤维细胞的基质层构建:
(1)取第8代***成纤维细胞,用含20%胎牛血清的DMEM配成密度为5×106个/mL细胞悬液;
(2)将细胞悬液和中性胶原支架中性胶原支架按照1:9的体积比例混匀,接种到Transwell小室中,置于37℃条件下培养2.5小时,然后在Transwell小室内外分别添加HVE-1培养液,37℃,5%CO2培养箱培养3天,形成***粘膜模型中的基质层;
上述HVE-1培养液按照以下方法配制而成:以DMEM和F12按体积比2:1混合的培养基为基础液,在其中添加10%胎牛血清,4.0μmol/mL的谷氨酰胺,20μg/mL腺嘌呤,10μg/mL胰岛素,1.2μg/mL 氢化可的松,加入75μg/mL维生素C,5ng/mL成纤维细胞生长因子,15ng/mL转铁蛋白;
E、***上皮细胞的接种:
(1)吸去基质层表面的培养基,将上述得到的***上皮细胞或A431细胞系用HVE-2培养液配制成2.0×106个/mL细胞悬液,接种到Transwell小室中的基质层上;
(2)将培养基更换成HVE-2培养液进行浸没式培养,37℃、5%CO2培养箱中培养4天;
上述HVE-2培养液按照以下方法配制而成:以DMEM和F12按体积比2:1混合的培养基为基础液,在其中添加5%胎牛血清,4.5μmol/mL的谷氨酰胺,22μg/mL腺嘌呤,10μg/mL胰岛素,1.5μg/mL 氢化可的松,6ng/mL成纤维细胞生长因子,12ng/mL转铁蛋白,1.5ng/mLEGF,0.2nmol/mL三碘甲状腺原氨酸,8nmol/mL异丙肾上腺素;
F、***粘膜模型的培养:
(1)吸去Transwell小室中培养物表面的HVE-2培养液,将Transwell小室提升至空气-液体界面进行气液面培养;
(2)将HVE-2培养液更换成HVE-3培养液, 37℃、5%CO2培养箱中培养10天,得到基质层中含有成纤维细胞、上皮层中含有***上皮细胞的双层***粘膜模型;
上述HVE-3培养液按照以下方法配制而成:以DMEM和F12按体积比2:1混合的培养基为基础液,在其中添加5%胎牛血清,3.0μmol/mL的谷氨酰胺,18μg/mL腺嘌呤,12μg/mL胰岛素,1.5μg/mL 氢化可的松,5.0ng/mL成纤维细胞生长因子,15ng/mL转铁蛋白,3.5ng/mLEGF,0.25nmol/mL三碘甲状腺原氨酸,8nmol/mL异丙肾上腺素,60μg/mL维生素C,1.2μmol/mL CaCl2。
实施例3
本实施例与实施例1的区别之处在于:
步骤A中所述无血清上皮细胞培养液以DMEM和F12按体积比3:1混合的培养基为基础液,在其中添加6.0μmol/mL的谷氨酰胺,5.0ng/mL EGF,30μg/mL BPE,15ng/mL胰岛素,2.0μg/mL 氢化可的松,0.4μmol/mL CaCl2。
实施例4
一种人***粘膜模型的体外构建方法,包括如下步骤:
A、外阴表皮样癌细胞系A431细胞培养:
重悬外阴表皮样癌细胞系A431细胞沉淀于含10%胎牛血清、2.0μmol/mL谷氨酰胺、0.1μmol/mL丙酮酸钠和4.5g/L高糖的DMEM培养液,37℃,5%CO2培养箱传代培养至第七代后用于进行上皮层的构建;
B、***成纤维细胞的分离和培养:
(1)将分离的***组织固有层放入200U/mL胶原酶消化液中,37℃消化3h,使用含10%胎牛血清的DMEM培养液终止消化,200目筛网过滤,离心去上清,收集***成纤维细胞;
(2)PBS清洗***成纤维细胞,重悬细胞沉淀于含10%新生牛血清的DMEM培养液中,接种于T75培养瓶培养,37℃,5%CO2条件下传代培养至第五代后用于进行***粘膜模型中基质层的构建;
C、中性胶原支架的制备:
称量胶原置于0.1%醋酸溶液中配制成10mg/mL的胶原溶液,待完全溶解后置于冰上,将胶原溶液和含10%胎牛血清DEME培养液按体积比1:8混匀,加入0.1M的NaOH溶液调节PH至7.4;
D、含成纤维细胞的基质层构建:
(1)取第10代***成纤维细胞,用含20%胎牛血清的DMEM配成密度为8×106个/mL细胞悬液;
(2)将细胞悬液和中性胶原支架按照1:9的体积比例混匀,接种到Transwell小室中,置于37℃条件下培养3小时,然后在Transwell小室内外分别添加HVE-1培养液,37℃,5%CO2培养箱培养4天,形成***粘膜模型中的基质层;
上述HVE-1培养液按照以下方法配制而成:以DMEM和F12按体积比3:1混合的培养基为基础液,在其中添加10%胎牛血清,在其中添加6.0μmol/mL的谷氨酰胺,30μg/mL腺嘌呤,15μg/mL胰岛素,2.0μg/mL 氢化可的松,加入100μg/mL维生素C,10ng/mL成纤维细胞生长因子,20ng/mL转铁蛋白;
E、外阴表皮样癌细胞系A431细胞的接种:
(1)吸去基质层表面的培养基,将上述得到的A431细胞系用HVE-2培养液配制成5.0×106个/mL细胞悬液,接种到Transwell小室中的基质层上;
(2)将培养基更换成HVE-2培养液进行浸没式培养,37℃、5%CO2培养箱中培养4天;
上述HVE-2培养液按照以下方法配制而成:以DMEM和F12按体积比3:1混合的培养基为基础液,在其中添加5%胎牛血清,6.0μmol/mL的谷氨酰胺,30μg/mL腺嘌呤,15μg/mL胰岛素,2.0μg/mL 氢化可的松,2.0ng/mL成纤维细胞生长因子,20ng/mL转铁蛋白,2.0ng/mLEGF,0.4nmol/mL三碘甲状腺原氨酸,10nmol/mL异丙肾上腺素;
F、***粘膜模型的培养:
(1)吸去Transwell小室中培养物表面的HVE-2培养液,将Transwell小室提升至空气-液体界面进行气液面培养;
(2)将HVE-2培养液更换成HVE-3培养液, 37℃、5%CO2培养箱中培养8~12天,得到基质层中含有成纤维细胞、上皮层中含有外阴表皮样癌细胞系A431的双层***粘膜模型;
上述HVE-3培养液按照以下方法配制而成:以DMEM和F12按体积比3:1混合的培养基为基础液,在其中添加5%胎牛血清,6.0μmol/mL的谷氨酰胺,30μg/mL腺嘌呤,15μg/mL胰岛素,2.0μg/mL 氢化可的松,10ng/mL成纤维细胞生长因子,20ng/mL转铁蛋白,5.0ng/mLEGF,0.4nmol/mL三碘甲状腺原氨酸,10nmol/mL异丙肾上腺素,100μg/mL维生素C,1.5μmol/mL CaCl2。
实施例5
本实施例与实施例4的区别在于:
步骤A:外阴表皮样癌细胞系A431细胞培养:
重悬外阴表皮样癌细胞系A431细胞沉淀于含10%胎牛血清、4.5μmol/mL谷氨酰胺、0.5μmol/mL丙酮酸钠和4.5g/L高糖的DMEM培养液,37℃,5%CO2培养箱传代培养至第七代后用于进行上皮层的构建。
实施例6
本实施例与实施例4的区别在于:
步骤A:外阴表皮样癌细胞系A431细胞培养:
重悬外阴表皮样癌细胞系A431细胞沉淀于含10%胎牛血清、6.0μmol/mL谷氨酰胺、1μmol/mL丙酮酸钠和4.5g/L高糖的DMEM培养液,37℃,5%CO2培养箱传代培养至第七代后用于进行上皮层的构建。
由图1A和图1B可知,使用正常人***上皮细胞培养构建的***粘膜模型具有与正常***组织相似的结构特征,其上皮层主要分为三层,最上层为分化成熟的角质层,中间为移行层,最下层为基底层,基质层为包含有成纤维细胞的胶原层,表明本发明所建立的3D***粘膜模型可以在体外正常分化,适用于人正常生殖道上皮的生理学研究、药物毒性安全性评价和体外***刺激性实验。
由图2A和图2B可知,使用外阴表皮样癌细胞系A431所构建的***粘膜模型其上皮层不能分化出成熟的角质层,但是该模型具有一定的屏障功能,且构建周期短,种子细胞来源广泛,性能比较稳定,能够满足体外***刺激性实验的基本要求。
以上所述,仅为本申请的具体实施方式,但本申请的保护范围并不局限于此,任何在本申请揭露的技术范围内的变化或替换,都应涵盖在本申请的保护范围之内。因此,本申请的保护范围应以所述权利要求的保护范围为准。
Claims (10)
1.一种人***粘膜模型的体外构建方法,其特征在于,包括如下步骤:
A、含成纤维细胞的基质层构建:
(1)取第5~10代***成纤维细胞,用含20%胎牛血清的DMEM配成细胞悬液;
(2)将细胞悬液和中性胶原支架按照1:9的体积比例混匀,接种到细胞培养小室中首次培养,然后在小室内外分别添加HVE-1培养液,二次培养后形成***粘膜模型中的基质层;
所述HVE-1培养液按照以下方法配制而成:以DMEM和F12按体积比1~3:1混合的培养基为基础液,在其中添加10%胎牛血清,添加2.0~6.0μmol/mL的谷氨酰胺,15~30μg/mL腺嘌呤,5~15μg/mL胰岛素,0.5~2.0μg/mL 氢化可的松,25~100μg/mL维生素C,1~10ng/mL成纤维细胞生长因子和5~20ng/mL转铁蛋白;
B、细胞的接种:
吸去基质层表面的HVE-1培养液,将***上皮细胞或外阴表皮样癌细胞系A431细胞用HVE-2培养液配制成细胞悬液,接种到细胞培养小室中的基质层上,将培养基更换成HVE-2培养液进行浸没式培养;
所述HVE-2培养液按照以下方法配制而成:以DMEM和F12按体积比1~3:1混合的培养基为基础液,在其中添加5%胎牛血清,2.0~6.0μmol/mL的谷氨酰胺,15~30μg/mL腺嘌呤,5~15μg/mL胰岛素,0.5~2.0μg/mL 氢化可的松,1~10ng/mL成纤维细胞生长因子,5~20ng/mL转铁蛋白,0.5~2.0ng/mL 表皮生长因子,0.1~0.4nmol/mL三碘甲状腺原氨酸和5~10nmol/mL异丙肾上腺素;
C、***粘膜模型的培养:
吸去培养物表面的HVE-2培养液,将细胞培养小室提升至空气-液体界面进行气液面培养;将培养基更换成HVE-3培养液,培养后得到双层***粘膜模型;
所述HVE-3培养液按照以下方法配制而成:以DMEM和F12按体积比1~3:1混合的培养基为基础液,在其中添加5%胎牛血清,2.0~6.0μmol/mL的谷氨酰胺,15~30μg/mL腺嘌呤,5~15μg/mL胰岛素,0.5~2.0μg/mL氢化可的松,1~10ng/mL成纤维细胞生长因子,5~20ng/mL转铁蛋白,2.0~5.0ng/mL 表皮生长因子,0.1~0.4nmol/mL三碘甲状腺原氨酸,5~10nmol/mL异丙肾上腺素,25~100μg/mL维生素C和1.0~1.5μmol/mL CaCl2。
2.如权利要求1所述的构建方法,其特征在于,步骤A中***成纤维细胞的分离和培养步骤如下:
(1)将分离的***组织固有层放入胶原酶消化液中消化,然后使用含10%胎牛血清的DMEM培养液终止消化,过滤后离心去上清,收集***成纤维细胞;
(2)PBS清洗***成纤维细胞,重悬细胞沉淀于含10%新生牛血清的DMEM培养液中,传代培养至第五代后用于进行***粘膜模型中基质层的构建。
3.如权利要求1所述的构建方法,其特征在于,步骤B中***上皮细胞的分离和培养步骤如下:
(1)将人体***组织置于培养皿中, PBS溶液清洗6次,除去粘膜下组织,将组织块切块加入分散酶中消化;
(2)眼科镊分离出***粘膜上皮层和固有层,将***粘膜上皮层放入胰酶-EDTA中消化;
(3)使用含10%胎牛血清的DMEM培养液终止消化,过滤后离心去上清,收集***粘膜上皮细胞;
(4)PBS清洗***粘膜上皮细胞,重悬细胞沉淀于无血清上皮细胞培养液中,获得分离成单个细胞的***上皮细胞悬液,传代培养至第二代得到纯度达95%以上的***上皮细胞。
4.如权利要求1所述的构建方法,其特征在于,步骤B中外阴表皮样癌细胞系A431细胞的培养过程为:重悬外阴表皮样癌细胞系A431细胞沉淀于含10%胎牛血清、2.0~6.0μmol/mL谷氨酰胺、0.1~1μmol/mL丙酮酸钠和4.5g/L高糖的DMEM培养液,传代培养至第七代后用于进行上皮层的构建。
5.如权利要求1所述的构建方法,其特征在于,步骤A步骤A中中性胶原支架的制备方法为:称量胶原置于0.1%醋酸溶液中配制成4~10mg/mL的胶原溶液,待完全溶解后置于冰上,将胶原溶液和含10%胎牛血清DEME培养液按体积比1:8混匀,加入0.1M的NaOH溶液调节PH至7.2~7.4。
6.如权利要求1所述的构建方法,其特征在于,步骤A中首次培养温度为37℃,首次培养时间为2~3小时,二次培养温度为37℃,二次培养时间为2~4天。
7.如权利要求1所述的构建方法,其特征在于,步骤B中培养温度为37℃,培养时间为4天。
8.如权利要求1所述的构建方法,其特征在于,步骤C中培养温度为37℃,培养时间为8~12天。
9.如权利要求2或3所述的构建方法,其特征在于,所述PBS溶液中含有100U/mL青霉素和100U/mL链霉素。
10.如权利要求3所述的构建方法,其特征在于,所述EDTA浓度为0.2mg/mL。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110356820.4A CN113293126B (zh) | 2021-04-01 | 2021-04-01 | 一种人***粘膜模型的体外构建方法 |
CN202110575428.9A CN113862214A (zh) | 2021-04-01 | 2021-04-01 | 用于构建人***粘膜模型培养基的配制方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110356820.4A CN113293126B (zh) | 2021-04-01 | 2021-04-01 | 一种人***粘膜模型的体外构建方法 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110575428.9A Division CN113862214A (zh) | 2021-04-01 | 2021-04-01 | 用于构建人***粘膜模型培养基的配制方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113293126A CN113293126A (zh) | 2021-08-24 |
CN113293126B true CN113293126B (zh) | 2023-11-07 |
Family
ID=77319355
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110356820.4A Active CN113293126B (zh) | 2021-04-01 | 2021-04-01 | 一种人***粘膜模型的体外构建方法 |
CN202110575428.9A Withdrawn CN113862214A (zh) | 2021-04-01 | 2021-04-01 | 用于构建人***粘膜模型培养基的配制方法 |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110575428.9A Withdrawn CN113862214A (zh) | 2021-04-01 | 2021-04-01 | 用于构建人***粘膜模型培养基的配制方法 |
Country Status (1)
Country | Link |
---|---|
CN (2) | CN113293126B (zh) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5518915A (en) * | 1986-04-18 | 1996-05-21 | Advanced Tissue Sciences, Inc. | Three-Dimensional mucosal cell and tissue culture system |
CN106635962A (zh) * | 2017-01-20 | 2017-05-10 | 李晖 | 一种人正常***上皮3d分化培养物模型的构建方法与用途 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6943021B2 (en) * | 2002-06-07 | 2005-09-13 | Mattek Corporation | Three dimensional vaginal tissue model containing immune cells |
-
2021
- 2021-04-01 CN CN202110356820.4A patent/CN113293126B/zh active Active
- 2021-04-01 CN CN202110575428.9A patent/CN113862214A/zh not_active Withdrawn
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5518915A (en) * | 1986-04-18 | 1996-05-21 | Advanced Tissue Sciences, Inc. | Three-Dimensional mucosal cell and tissue culture system |
CN106635962A (zh) * | 2017-01-20 | 2017-05-10 | 李晖 | 一种人正常***上皮3d分化培养物模型的构建方法与用途 |
Also Published As
Publication number | Publication date |
---|---|
CN113862214A (zh) | 2021-12-31 |
CN113293126A (zh) | 2021-08-24 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Burwen et al. | Secretory function of lactating mouse mammary epithelial cells cultured on collagen gels | |
US5834312A (en) | Process and media for the growth of human epithelia | |
Chuman et al. | Continuous growth of proximal tubular kidney epithelial cells in hormone-supplemented serum-free medium. | |
US7037721B1 (en) | Protein-free defined media for the growth of normal human keratinocytes | |
CN109749997B (zh) | 一种角膜缘干细胞无血清培养基及其培养方法 | |
Chopra et al. | Propagation of differentiating normal human tracheobronchial epithelial cells in serum‐free medium | |
CN101705207B (zh) | 一种羊水细胞培养基 | |
CN114107173A (zh) | 一种血管化胰岛微器官及其构建方法 | |
CN113293126B (zh) | 一种人***粘膜模型的体外构建方法 | |
CN113621554B (zh) | 采用同一无血清培养基的表皮组织简易制备工艺及其保存 | |
CN107312744B (zh) | 一种含血清的口腔黏膜上皮细胞培养液 | |
WO2019029084A1 (zh) | 3d打印人造子宫内膜及其制备方法和应用 | |
CN114134100A (zh) | 用于构建人***粘膜模型培养基的配制方法 | |
CN113862215A (zh) | 用于构建人***粘膜模型培养基的配制方法 | |
CN112760289A (zh) | 一种乳腺癌类器官专用培养基及3d培养方法 | |
EP1690929A1 (en) | Regeneration treatment system | |
WO2007008521A1 (en) | Stage specific follicle maturation systems | |
CN113106066B (zh) | 一种肉瘤细胞培养基以及采用该培养基体外生产Matrigel原液的方法 | |
CN107312745B (zh) | 一种无血清的上皮细胞培养液 | |
Schumann et al. | Isolation, characterization, and long-term culture of fetal bovine tracheal epithelial cells | |
CN115820548A (zh) | 一种动物组织来源的凋亡囊泡的制备方法及其应用 | |
McAteer et al. | Polarized epithelial cysts in vitro: a review of cell and explant culture systems that exhibit epithelial cyst formation | |
Kawamura et al. | Primary culture of epithelial cells derived from the rat ventral prostate: formation of three‐dimensional acinus‐like structure in collagen gel | |
Simpson et al. | Characterization of endocrine-rich monolayers of human fetal pancreas that display reduced immunogenicity | |
EP3881853A1 (en) | Human allogeneic liver-derived progenitor cells for use in the treatment of inflammatory and infectious lung diseases and systemic inflammation |
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 |