CN112760289B - 一种乳腺癌类器官专用培养基及3d培养方法 - Google Patents
一种乳腺癌类器官专用培养基及3d培养方法 Download PDFInfo
- Publication number
- CN112760289B CN112760289B CN202110095023.5A CN202110095023A CN112760289B CN 112760289 B CN112760289 B CN 112760289B CN 202110095023 A CN202110095023 A CN 202110095023A CN 112760289 B CN112760289 B CN 112760289B
- Authority
- CN
- China
- Prior art keywords
- culture medium
- breast cancer
- culture
- days
- organoids
- 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
- 210000002220 organoid Anatomy 0.000 title claims abstract description 54
- 239000001963 growth medium Substances 0.000 title claims abstract description 49
- 206010006187 Breast cancer Diseases 0.000 title claims abstract description 36
- 208000026310 Breast neoplasm Diseases 0.000 title claims abstract description 36
- 238000012136 culture method Methods 0.000 title claims abstract description 17
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 claims abstract description 22
- 210000001519 tissue Anatomy 0.000 claims abstract description 14
- 102000009016 Cholera Toxin Human genes 0.000 claims abstract description 11
- 108010049048 Cholera Toxin Proteins 0.000 claims abstract description 11
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 claims abstract description 11
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims abstract description 11
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 claims abstract description 11
- 229930182566 Gentamicin Natural products 0.000 claims abstract description 11
- 229960005309 estradiol Drugs 0.000 claims abstract description 11
- 229960002518 gentamicin Drugs 0.000 claims abstract description 11
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 claims abstract description 10
- 239000000654 additive Substances 0.000 claims abstract description 10
- 230000000996 additive effect Effects 0.000 claims abstract description 10
- 238000012258 culturing Methods 0.000 claims abstract description 9
- 108010082117 matrigel Proteins 0.000 claims abstract description 7
- 238000001914 filtration Methods 0.000 claims abstract description 5
- 238000002156 mixing Methods 0.000 claims abstract description 5
- 239000013049 sediment Substances 0.000 claims abstract description 3
- 210000004027 cell Anatomy 0.000 claims description 25
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 14
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 14
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 12
- 229940098773 bovine serum albumin Drugs 0.000 claims description 12
- 210000004263 induced pluripotent stem cell Anatomy 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 8
- 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 8
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 7
- 102400001368 Epidermal growth factor Human genes 0.000 claims description 7
- 101800003838 Epidermal growth factor Proteins 0.000 claims description 7
- 229940116977 epidermal growth factor Drugs 0.000 claims description 7
- 102000048238 Neuregulin-1 Human genes 0.000 claims description 4
- 108090000556 Neuregulin-1 Proteins 0.000 claims description 4
- DBLXOVFQHHSKRC-UHFFFAOYSA-N ethanesulfonic acid;2-piperazin-1-ylethanol Chemical compound CCS(O)(=O)=O.OCCN1CCNCC1 DBLXOVFQHHSKRC-UHFFFAOYSA-N 0.000 claims description 3
- 239000008188 pellet Substances 0.000 claims description 2
- 230000010261 cell growth Effects 0.000 claims 1
- 210000001339 epidermal cell Anatomy 0.000 claims 1
- 239000003102 growth factor Substances 0.000 claims 1
- 230000002934 lysing effect Effects 0.000 claims 1
- 206010028980 Neoplasm Diseases 0.000 abstract description 15
- MVCOAUNKQVWQHZ-UHFFFAOYSA-N doramapimod Chemical compound C1=CC(C)=CC=C1N1C(NC(=O)NC=2C3=CC=CC=C3C(OCCN3CCOCC3)=CC=2)=CC(C(C)(C)C)=N1 MVCOAUNKQVWQHZ-UHFFFAOYSA-N 0.000 abstract description 10
- 229950005521 doramapimod Drugs 0.000 abstract description 10
- 238000000338 in vitro Methods 0.000 abstract description 4
- 239000002244 precipitate Substances 0.000 abstract description 3
- 230000001112 coagulating effect Effects 0.000 abstract description 2
- 238000005336 cracking Methods 0.000 abstract description 2
- 239000013618 particulate matter Substances 0.000 abstract 1
- 238000010586 diagram Methods 0.000 description 19
- 230000000052 comparative effect Effects 0.000 description 10
- 210000004881 tumor cell Anatomy 0.000 description 9
- 230000000694 effects Effects 0.000 description 6
- 239000002609 medium Substances 0.000 description 5
- 238000012364 cultivation method Methods 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 230000008859 change Effects 0.000 description 3
- OLIIUAHHAZEXEX-UHFFFAOYSA-N N-(6-fluoro-1H-indazol-5-yl)-6-methyl-2-oxo-4-[4-(trifluoromethyl)phenyl]-3,4-dihydro-1H-pyridine-5-carboxamide Chemical compound C1C(=O)NC(C)=C(C(=O)NC=2C(=CC=3NN=CC=3C=2)F)C1C1=CC=C(C(F)(F)F)C=C1 OLIIUAHHAZEXEX-UHFFFAOYSA-N 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000012679 serum free medium Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 1
- 206010070863 Toxicity to various agents Diseases 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 230000008611 intercellular interaction Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000036210 malignancy Effects 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
- 230000001338 necrotic effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 238000013414 tumor xenograft model Methods 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
- C12N5/0693—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
- 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
- C12N5/0631—Mammary 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
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/01—Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
-
- 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
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- 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
- C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
-
- 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
- C12N2501/15—Transforming growth factor beta (TGF-β)
-
- 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
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/39—Steroid hormones
- C12N2501/392—Sexual steroids
-
- 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
- C12N2501/40—Regulators of development
- C12N2501/405—Cell cycle regulated proteins, e.g. cyclins, cyclin-dependant kinases
-
- 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.]
- C12N2501/724—Glycosyltransferases (EC 2.4.)
-
- 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
- C12N2501/998—Proteins not provided for elsewhere
-
- 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
- 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
- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
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)
- Oncology (AREA)
- Dermatology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供一种乳腺癌类器官专用培养基及3D培养方法,该培养基包括基础培养基和特异性添加因子;所述特异性添加因子包括GSK429286A;Doramapimod;A83‑01;EGF;β‑estradiol;Cholera Toxin;FGF2;HEPES;B27;GlutaMAX;ITS;Gentamicin;Neuregulin‑1;BSA。培养方法包括:将分离的乳腺癌组织进行预处理后消化并过滤,得到细胞沉淀;将细胞沉淀裂解,然后与matrigel胶混匀并接种,待混合胶凝固后加入上述培养基,并于37℃、5%CO2浓度下培养7‑14天。本发明的培养基可实现乳腺癌类器官的快速生长,并可长期稳定培养,形成的肿瘤类器官细胞球球形规则,且大小较为均一,能在体外很好地保留患者肿瘤组织的异质性。
Description
技术领域
本发明属于细胞培养技术领域,具体涉及一种乳腺癌类器官专用培养基及3D培养方法。
背景技术
乳腺癌是全球女性最常见的恶性肿瘤之一,也是导致女性癌症患者死亡的第二大手。近年来,随着我国经济的发展和人们生活方式的改变,我国乳腺癌的发病率逐年升高,并且患者呈现低龄化趋势。目前,学者们主要使用传统的肿瘤细胞株模型(CCL)和患者来源的肿瘤异种移植模型(PDTX)来研究乳腺癌的发生发展情况。虽然两种模型为乳腺癌的研究做出了巨大贡献,但均存在一定的局限性,如CCL模型可能会出现交污染、基因型会发生改变以及很难永久性增殖等,PDTX模型的瘤移植成功率低、建立所需的时间长、受体缺乏免疫能力以及不能进行高通量药物筛选等,这些缺点可能导致实验结果与肿瘤在人体内的实际情况出现偏差,因此迫切希望一种更加优秀的模型出现。
类器官是由多能干细胞或器官祖细胞分化,并自组装成有结构和功能的完整哺乳动物器官,类器官技术在研究广泛的对象方面潜力巨大,包括发育生物学、疾病病理学、细胞生物学、再生机制、精准医疗以及药物毒性和药效试验等。对于这些应用以及其他应用,类器官培养实现了对现有2D培养方法和动物模型系统的高信息量的互补。
发明内容
有鉴于此,本发明旨在提供一种乳腺癌类器官专用培养基及3D培养方法以解决上述问题。本发明的技术方案为:
第一个方面,本发明提供一种乳腺癌类器官专用培养基,包括基础培养基和特异性添加因子;所述特异性添加因子包括GSK429286A;达马莫德(Doramapimod);A83-01;表皮细胞生长因子(EGF);β-雌二醇(β-estradiol);霍乱毒素(Cholera Toxin);碱性成纤维细胞生长因子(FGF2);4-羟乙基哌嗪乙磺酸(HEPES);B27;GlutaMAX;诱导性多功能干细胞(ITS);庆大霉素(Gentamicin);神经调节因子1(Neuregulin-1);牛血清白蛋白(BSA)。
进一步地,所述特异性添加因子包括以下终浓度的组分:GSK429286A:5-20μM;Doramapimod:5-20μM;A83-01:0.5-1μM;EGF:50-500ng/ml;β-estradiol:2-10nM;CholeraToxin:10-30ng/ml;FGF2:1-5μM;HEPES:10-30mM;B27:1-5X;GlutaMAX:1-2X;ITS:0.1-1X;Gentamicin:0.5-50μg/ml;Neuregulin-1:2-10nM;牛血清白蛋白(BSA):1-10mg/ml。
进一步地,所述培养基还包括重组蛋白。
优选地,所述重组蛋白为TN-C重组蛋白,其在所述培养基中的终浓度为50-100ng/ml。
更优选地,所述特异性添加因子包括以下终浓度的组分:GSK429286A:10-15μM;Doramapimod:6-8μM;A83-01:0.7-0.8μM;EGF:150-200ng/ml;β-estradiol:4-6nM;CholeraToxin:10-15ng/ml;FGF2:1-5μM;HEPES:15-20mM;B27:1-5X;GlutaMAX:1-2X;ITS:0.5-1X;Gentamicin:20-30μg/ml;Neuregulin-1:5-6nM;牛血清白蛋白(BSA):4-5mg/ml;TN-C重组蛋白:70-80ng/ml。
进一步地,所述基础培养基为无血清培养基。
优选地,所述无血清培养基为DMEM/F12(1:1)减血清培养基。
进一步地,所述培养基的制备方法为:将特异性添加因子加入到基础培养基中混合均匀既得。
第二个方面,本发明提供一种乳腺癌类器官3D培养方法,包括以下步骤:
1)将分离的乳腺癌组织进行预处理后消化并过滤,得到细胞沉淀;
2)将细胞沉淀裂解,然后与matrigel胶混匀并接种,待混合胶凝固后加入上述培养基,并于37℃、5%CO2浓度下培养7-14天,每间隔2-3天更换一次培养基,即得。
第三个方面,本发明提供一种乳腺癌类器官,是采用上述培养方法获得。
本发明的有益效果是:
①本发明的培养基可实现乳腺癌类器官的快速生长,并可长期稳定培养,形成的肿瘤类器官细胞球形规则,且大小较为均一,能在体外很好地保留患者肿瘤组织的异质性。
②本发明的培养方法操作简单,人员操作影响较少,稳定性高。
附图说明
图1为本发明实施例1中培养1天的肿瘤细胞的状态图。
图2为本发明实施例1中培养4天的肿瘤细胞的状态图。
图3为本发明实施例1中培养7天的肿瘤细胞的状态图。
图4为本发明实施例1中培养10天的肿瘤细胞的状态图。
图5为本发明实施例1获得的类器官的形貌图。
图6为本发明实施例2获得的类器官的形貌图。
图7为本发明实施例3获得的类器官的形貌图。
图8为本发明实施例4获得的类器官的形貌图。
图9为本发明实施例5获得的类器官的形貌图。
图10为本发明实施例6中长期稳定培养30天的类器官的状态图。
图11为本发明实施例6中长期稳定培养60天的类器官的状态图。
图12为本发明实施例6中长期稳定培养90天的类器官的状态图。
图13为本发明对比例1中培养5天的肿瘤细胞的状态图。
图14为本发明对比例1中培养10天的肿瘤细胞的状态图。
图15为本发明对比例2中培养14天的肿瘤细胞的状态图。
图16为本发明对比例3中培养14天的肿瘤细胞的状态图。
具体实施方式
在本发明的描述中,需要说明的是,实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
下面结合附图和具体的实施例对本发明做进一步详细说明,所述是对本发明的解释而不是限定。
实施例1
本实施例提供一种乳腺癌类器官专用培养基及3D培养方法,该培养基包括基础培养基和特异性添加因子;所述特异性添加因子包括以下终浓度的组分:GSK429286A:10μM;Doramapimod:8μM;A83-01:0.8μM;EGF:150ng/ml;β-estradiol:5nM;Cholera Toxin:12ng/ml;FGF2:2μM;HEPES:20mM;B27:1X;GlutaMAX:2X;ITS:0.5X;Gentamicin:25μg/ml;Neuregulin-1:6nM;BSA:4mg/ml;TN-C重组蛋白:70ng/ml。培养方法包括:将取出的乳腺癌组织用生理盐水清洗后,在冰上剪碎,加入10ml胰酶重悬,转移至37℃,220rpm摇床消化10分钟,消化完成后加入5ml的DMEM/F12终止消化。用70μM的细胞滤网过滤细胞悬液,1200rpm,离心5分钟,加入5ml的红细胞裂解液重悬沉淀,用5ml的DMEM/F12终止裂解后离心,去除上清,细胞计数,混合matrigel,20000个细胞每滴,滴于24孔板中,放置于37℃,5%的CO2培养箱中,待matrigel胶凝固后,加入上述专用培养基于37℃、5%CO2浓度下培养10天,每间隔2-3天更换一次培养基,即得。图1~5提供了培养1天~10天的细胞状态变化图以及最终获得的类器官的形貌图,可见,本实施例的培养基可实现乳腺癌类器官的快速生长,形成的肿瘤类器官细胞球形规则,且大小较为均一,能在体外很好地保留患者肿瘤组织的异质性。
实施例2
本实施例提供一种乳腺癌类器官专用培养基及3D培养方法,该培养基包括基础培养基和特异性添加因子;所述特异性添加因子包括以下终浓度的组分:GSK429286A:15μM;Doramapimod:6μM;A83-01:0.7μM;EGF:200ng/ml;β-estradiol:4nM;Cholera Toxin:15ng/ml;FGF2:5μM;HEPES:15mM;B27:5X;GlutaMAX:1X;ITS:1X;Gentamicin:30μg/ml;Neuregulin-1:5nM;BSA:5mg/ml;TN-C重组蛋白:80ng/ml。培养方法同实施例1。培养7天获得的类器官形貌图如图6所示,形成的肿瘤类器官细胞球形规则,且直径可超200μM,活性较好。
实施例3
本实施例提供一种乳腺癌类器官专用培养基及3D培养方法,该培养基包括基础培养基和特异性添加因子;所述特异性添加因子包括以下终浓度的组分:GSK429286A:5μM;Doramapimod:15μM;A83-01:0.5μM;EGF:500ng/ml;β-estradiol:2nM;Cholera Toxin:25ng/ml;FGF2:3μM;HEPES:10mM;B27:3X;GlutaMAX:1X;ITS:0.1X;Gentamicin:10μg/ml;Neuregulin-1:2nM;BSA:10mg/ml;TN-C重组蛋白:100ng/ml。培养方法同实施例1。培养12天获得的类器官形貌图如图7所示,形成的肿瘤类器官细胞球形规则,且直径较大,活性较好。
实施例4
本实施例提供一种乳腺癌类器官专用培养基及3D培养方法,该培养基包括基础培养基和特异性添加因子;所述特异性添加因子包括以下终浓度的组分:GSK429286A:20μM;Doramapimod:5μM;A83-01:1μM;EGF:50ng/ml;β-estradiol:10nM;Cholera Toxin:10ng/ml;FGF2:1μM;HEPES:30mM;B27:5X;GlutaMAX:2X;ITS:0.3X;Gentamicin:50μg/ml;Neuregulin-1:10nM;BSA:1mg/ml;TN-C重组蛋白:100ng/ml。培养方法同实施例1。培养9天获得的类器官形貌图如图8所示,形成的肿瘤类器官数量较多,活性较好。
实施例5
本实施例提供一种乳腺癌类器官专用培养基及3D培养方法,该培养基与实施例1的区别在于:特异性添加因子中没有TN-C重组蛋白。培养方法同实施例1。培养14天获得的类器官形貌图如图9所示,表明在未加入TN-C重组蛋白的前提下,虽然也可以获得类器官,但细胞活性较比前几个实施例的都要差,且无直径大于50μM的类器官形成。
实施例6
本实施例提供一种乳腺癌类器官的培养方法,是采用实施例1的培养基,该培养方法包括以下步骤:
1)将取出的乳腺癌组织用生理盐水清洗后,在冰上剪碎,加入10ml胰酶重悬,转移至37℃,220rpm摇床消化10分钟,消化完成后加入5ml的DMEM/F12终止消化。
2)用70μM的细胞滤网过滤消化完的细胞悬液,1200rpm,离心5分钟,收集沉淀;
3)加入5ml的红细胞裂解液重悬沉淀,用5ml的DMEM/F12终止裂解后离心,去除上清,细胞计数;
4)计数完成后,将细胞沉淀混合matrigel,20000个细胞每滴,滴于24孔板中,放置于37℃,5%的CO2培养箱中,待matrigel胶凝固后,加入上述专用培养基于37℃、5%CO2浓度下培养30,60,90天,每间隔2-3天更换一次培养基,即得。图10~12提供了在30/60/90天获得的类器官的状态图,所形成的类器官结构规则,形态均匀,表明采用本发明的培养基可以对该类器官进行长期稳定培养。
对比例1
本对比例提供一种乳腺癌类器官的培养方法,该培养基与实施例1的区别在于:采用的是现有普通培养基(DMEM+10%FBS),并且不含有TN-C重组蛋白,培养过程同实施例1。图13和14提供了培养5天和10天的细胞状态图,乳腺癌组织细胞在培养过程中细胞生长缓慢且容易附着在培养皿底部,无法形成有球形结构的、多细胞成份的类器官。
对比例2
本对比例中提供的培养基中减去GSK429286A因子,其他同实施例1。
使用上述培养基按照实施例1的方法培养14天,获得的类器官状态如图15所示,结果为:减去GSK429286A因子后,细胞团连接松散活性较差,内部脱落坏死细胞增多。
对比例3
本对比例中提供的培养基中减去Doramapimod因子,其他同实施例1。
使用上述培养基按照实施例1的方法培养14天,获得的类器官状态如图16所示,结果为:减去Doramapimod因子后,细胞团直径普遍较小,且细胞间相互作用较差。
综上,本发明的培养基可实现乳腺癌类器官的快速生长,并可长期稳定培养,形成的肿瘤类器官细胞球形规则,且类器官直径较大,活性较好,能在体外很好地保留患者肿瘤组织的异质性。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (3)
1.一种乳腺癌类器官专用培养基,其特征在于:由基础培养基和特异性添加因子组成;所述特异性添加因子由以下终浓度的组分组成:GSK429286A:5-20μM;达马莫德:5-20μM;A83-01:0.5-1μM;表皮细胞生长因子:50-500ng/ml;β-雌二醇:2-10nM;霍乱毒素:10-30ng/ml;碱性成纤维细胞生长因子:1-5μM;4-羟乙基哌嗪乙磺酸:10-30mM;B27:1-5X;GlutaMAX:1-2X;诱导性多功能干细胞:0.1-1X;庆大霉素:0.5-50μg/ml;神经调节因子1:2-10nM;牛血清白蛋白:1-10mg/ml;TN-C重组蛋白:50-100ng/ml;所述基础培养基为DMEM/F12 1:1减血清培养基。
2.根据权利要求1所述的一种乳腺癌类器官专用培养基,其特征在于:所述特异性添加因子由以下终浓度的组分组成:GSK429286A:10-15μM;达马莫德:6-8μM;A83-01:0.7-0.8μM;表皮细胞生长因子:150-200ng/ml;β-雌二醇:4-6nM;霍乱毒素:10-15ng/ml;碱性成纤维细胞生长因子:1-5μM;4-羟乙基哌嗪乙磺酸:15-20mM;B27:1-5X;GlutaMAX:1-2X;诱导性多功能干细胞:0.5-1X;庆大霉素:20-30μg/ml;神经调节因子1:5-6nM;牛血清白蛋白:4-5mg/ml;TN-C重组蛋白:70-80ng/ml。
3.一种乳腺癌类器官3D培养方法,其特征在于:包括以下步骤:
1)将分离的乳腺癌组织进行预处理后消化并过滤,得到细胞沉淀;
2)将细胞沉淀裂解,然后与matrigel胶混匀并接种,待混合胶凝固后加入权利要求1或2所述的培养基,并于37℃、5%CO2浓度下培养7-14天,每间隔2-3天更换一次所述培养基,即得。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110095023.5A CN112760289B (zh) | 2021-01-25 | 2021-01-25 | 一种乳腺癌类器官专用培养基及3d培养方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110095023.5A CN112760289B (zh) | 2021-01-25 | 2021-01-25 | 一种乳腺癌类器官专用培养基及3d培养方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112760289A CN112760289A (zh) | 2021-05-07 |
CN112760289B true CN112760289B (zh) | 2022-04-12 |
Family
ID=75707041
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110095023.5A Active CN112760289B (zh) | 2021-01-25 | 2021-01-25 | 一种乳腺癌类器官专用培养基及3d培养方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112760289B (zh) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113583961A (zh) * | 2021-07-28 | 2021-11-02 | 扈晖 | 一种乳腺癌类器官无血清专用培养基 |
CN115910214B (zh) * | 2022-10-13 | 2023-10-13 | 南京普恩瑞生物科技有限公司 | 一种利用肿瘤活组织生物样本库模拟临床试验评估抗肿瘤药物药效的方法及其应用 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB201715930D0 (en) * | 2017-09-30 | 2017-11-15 | Upside Biotechnologies Ltd | Cell Culture Medium |
CN111876386B (zh) * | 2020-08-10 | 2023-05-30 | 上海市第一人民医院 | 一种乳腺癌类器官的培养方法以及和肿瘤相关成纤维细胞的共培养方法 |
-
2021
- 2021-01-25 CN CN202110095023.5A patent/CN112760289B/zh active Active
Also Published As
Publication number | Publication date |
---|---|
CN112760289A (zh) | 2021-05-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN113388569B (zh) | 肝脏类器官的制备方法 | |
CN114317443B (zh) | 乳腺癌类器官培养液及其培养试剂组合和培养方法 | |
CN111808817B (zh) | 一种骨肉瘤类器官的培养方法及其骨肿瘤培养基 | |
CN112760289B (zh) | 一种乳腺癌类器官专用培养基及3d培养方法 | |
CN109609441A (zh) | 一种肾脏组织类器官3d培养的培养基及类器官培养方法 | |
CN108504625B (zh) | 一种小鼠成纤维细胞及其用途 | |
CN112760288B (zh) | 一种肺癌类器官专用培养基及培养方法 | |
CN115161283B (zh) | 一种用于肝门部胆管癌来源类器官定向分化和培养的组合物及应用 | |
US20130189232A1 (en) | Cellular therapeutic agent for incontinence or urine comprising stem cells originated from decidua or adipose | |
CN111534477B (zh) | 小鼠肺组织原代上皮干细胞球培养方法 | |
CN101821383A (zh) | 一种用于人成体原始间充质干细胞体外规模化培养的培养基、方法及获得的原始间充质干细胞及其应用 | |
CN116024159A (zh) | 构建鼠类皮肤类器官的方法 | |
CN107129966A (zh) | 一种含血清的角膜上皮细胞培养液 | |
CN118345040A (zh) | 用于培养结直肠癌类器官的新型培养基 | |
CN112481193A (zh) | 三维培养肠及肠癌组织类器官的标准化培养基及培养方法 | |
CN116590232A (zh) | 一种甲状腺癌类器官、培养基及培养方法 | |
CN113444679B (zh) | 一种人泪腺干细胞及其分化培养方法与应用 | |
CN112501119B (zh) | 一种垂体腺瘤类器官培养基及其应用 | |
Huang et al. | A novel primary culture method for rat choroidal epithelial cells | |
CN116536264A (zh) | 一种结肠癌类器官无血清专用培养基 | |
US20110250236A1 (en) | Stem cells derived from the carotid body and uses thereof | |
CN118240762B (zh) | 一种模拟微重力诱导原代癌细胞重编程的培养方法及其应用 | |
CN115232792B (zh) | 一种用于胸水来源类器官的培养基及培养方法 | |
CN117106724A (zh) | 一种h3k27m突变型脊髓胶质瘤细胞及其培养方法 | |
CN118384331A (zh) | 一种脱细胞基质水凝胶及其制备方法与应用 |
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 |