CN116875459A - Organ chip for constructing malignant tumor bone metastasis ecological niche, organ chip and application - Google Patents
Organ chip for constructing malignant tumor bone metastasis ecological niche, organ chip and application Download PDFInfo
- Publication number
- CN116875459A CN116875459A CN202310860325.6A CN202310860325A CN116875459A CN 116875459 A CN116875459 A CN 116875459A CN 202310860325 A CN202310860325 A CN 202310860325A CN 116875459 A CN116875459 A CN 116875459A
- Authority
- CN
- China
- Prior art keywords
- channel
- micro
- chip
- microchannel
- tumor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000000988 bone and bone Anatomy 0.000 title claims abstract description 75
- 206010027476 Metastases Diseases 0.000 title claims abstract description 58
- 230000009401 metastasis Effects 0.000 title claims abstract description 58
- 210000000056 organ Anatomy 0.000 title claims abstract description 33
- 201000011510 cancer Diseases 0.000 title claims abstract description 23
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 84
- 239000007788 liquid Substances 0.000 claims abstract description 41
- 230000005059 dormancy Effects 0.000 claims abstract description 30
- 230000004087 circulation Effects 0.000 claims abstract description 29
- 238000013461 design Methods 0.000 claims abstract description 29
- 239000001963 growth medium Substances 0.000 claims abstract description 26
- 230000003211 malignant effect Effects 0.000 claims abstract description 16
- 238000004113 cell culture Methods 0.000 claims abstract description 12
- 230000002792 vascular Effects 0.000 claims abstract description 7
- 230000007420 reactivation Effects 0.000 claims abstract description 6
- 210000003556 vascular endothelial cell Anatomy 0.000 claims abstract description 5
- 239000000017 hydrogel Substances 0.000 claims description 39
- 210000004027 cell Anatomy 0.000 claims description 36
- 230000024245 cell differentiation Effects 0.000 claims description 29
- 210000004881 tumor cell Anatomy 0.000 claims description 23
- 230000015572 biosynthetic process Effects 0.000 claims description 16
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 claims description 14
- 208000020816 lung neoplasm Diseases 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 13
- 241000251539 Vertebrata <Metazoa> Species 0.000 claims description 12
- 229910052588 hydroxylapatite Inorganic materials 0.000 claims description 12
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 11
- 210000004204 blood vessel Anatomy 0.000 claims description 11
- 210000003557 bones of lower extremity Anatomy 0.000 claims description 11
- 238000000338 in vitro Methods 0.000 claims description 11
- 201000005202 lung cancer Diseases 0.000 claims description 11
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims description 8
- 210000002997 osteoclast Anatomy 0.000 claims description 6
- 238000010146 3D printing Methods 0.000 claims description 5
- 108010010803 Gelatin Proteins 0.000 claims description 4
- 230000000694 effects Effects 0.000 claims description 4
- 239000008273 gelatin Substances 0.000 claims description 4
- 229920000159 gelatin Polymers 0.000 claims description 4
- 235000019322 gelatine Nutrition 0.000 claims description 4
- 235000011852 gelatine desserts Nutrition 0.000 claims description 4
- 238000002513 implantation Methods 0.000 claims description 4
- 238000012544 monitoring process Methods 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- 210000001243 pseudopodia Anatomy 0.000 claims description 4
- 230000033228 biological regulation Effects 0.000 claims description 3
- 210000004271 bone marrow stromal cell Anatomy 0.000 claims description 3
- 206010027452 Metastases to bone Diseases 0.000 claims description 2
- CERQOIWHTDAKMF-UHFFFAOYSA-M Methacrylate Chemical compound CC(=C)C([O-])=O CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 claims 2
- 206010061289 metastatic neoplasm Diseases 0.000 abstract description 6
- 239000007943 implant Substances 0.000 abstract description 3
- 238000012546 transfer Methods 0.000 abstract description 3
- 230000002093 peripheral effect Effects 0.000 abstract description 2
- 238000010586 diagram Methods 0.000 description 10
- 238000011160 research Methods 0.000 description 10
- 238000011282 treatment Methods 0.000 description 10
- 230000034994 death Effects 0.000 description 6
- 231100000517 death Toxicity 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 230000007246 mechanism Effects 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 238000003501 co-culture Methods 0.000 description 5
- 238000004891 communication Methods 0.000 description 5
- 239000000975 dye Substances 0.000 description 5
- 238000007639 printing Methods 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- KPKZJLCSROULON-QKGLWVMZSA-N Phalloidin Chemical compound N1C(=O)[C@@H]([C@@H](O)C)NC(=O)[C@H](C)NC(=O)[C@H](C[C@@](C)(O)CO)NC(=O)[C@H](C2)NC(=O)[C@H](C)NC(=O)[C@@H]3C[C@H](O)CN3C(=O)[C@@H]1CSC1=C2C2=CC=CC=C2N1 KPKZJLCSROULON-QKGLWVMZSA-N 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 3
- 210000001185 bone marrow Anatomy 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 238000004088 simulation Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- 239000012114 Alexa Fluor 647 Substances 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 description 2
- 108010009711 Phalloidine Proteins 0.000 description 2
- 101150074062 Tnfsf11 gene Proteins 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 229940041181 antineoplastic drug Drugs 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 230000017531 blood circulation Effects 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 210000004292 cytoskeleton Anatomy 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000002073 fluorescence micrograph Methods 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 208000037841 lung tumor Diseases 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 230000001394 metastastic effect Effects 0.000 description 2
- 230000002085 persistent effect Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000004393 prognosis Methods 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 230000000284 resting effect Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000003319 supportive effect Effects 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 230000017423 tissue regeneration Effects 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 208000021959 Abnormal metabolism Diseases 0.000 description 1
- 208000018084 Bone neoplasm Diseases 0.000 description 1
- 206010006895 Cachexia Diseases 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 241000197236 Chrysanthemoides monilifera Species 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 206010015866 Extravasation Diseases 0.000 description 1
- 208000037147 Hypercalcaemia Diseases 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000003076 Osteolysis Diseases 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 206010033892 Paraplegia Diseases 0.000 description 1
- 206010041549 Spinal cord compression Diseases 0.000 description 1
- 208000005250 Spontaneous Fractures Diseases 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000004097 bone metabolism Effects 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 238000002619 cancer immunotherapy Methods 0.000 description 1
- 230000004709 cell invasion Effects 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000013170 computed tomography imaging Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 230000036251 extravasation Effects 0.000 description 1
- 238000001125 extrusion Methods 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000000148 hypercalcaemia Effects 0.000 description 1
- 208000030915 hypercalcemia disease Diseases 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000008611 intercellular interaction Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 208000029791 lytic metastatic bone lesion Diseases 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000006371 metabolic abnormality Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 210000002220 organoid Anatomy 0.000 description 1
- 230000011164 ossification Effects 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- 230000000010 osteolytic effect Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000036407 pain Effects 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 230000002572 peristaltic effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 208000011581 secondary neoplasm Diseases 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 210000002536 stromal cell Anatomy 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 210000005167 vascular cell Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/14—Scaffolds; Matrices
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/16—Particles; Beads; Granular material; Encapsulation
-
- 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
- C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
-
- 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
- C12N2502/1311—Osteocytes, osteoblasts, odontoblasts
-
- 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/28—Vascular endothelial 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
- C12N2513/00—3D culture
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Sustainable Development (AREA)
- Dispersion Chemistry (AREA)
- Clinical Laboratory Science (AREA)
- Immunology (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- Hematology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Organ chip for constructing bone metastasis ecological niche of malignant tumor and application thereof. The early stage of bone metastasis of malignant tumor has ecological niche reconstruction of primary tumor to bone metastasis, and relates to dormancy and reactivation of metastatic tumor, including tumor dormancy ecological niche, perivascular ecological niche and malignant circulation ecological niche. The organ chip simulates an H-shaped vascular structure of a bone at a transfer part, adopts an H-shaped runner design, and implants vascular endothelial cells on the peripheral wall of the channel for simulating a vascular system and a perivascular ecological niche. The H-shaped design comprises a micro-channel and a cell culture chamber, wherein the micro-channel at least comprises a first micro-channel, a second micro-channel, a third micro-channel and a liquid outlet micro-channel, the first micro-channel, the second micro-channel, the third micro-channel and the liquid outlet micro-channel are sequentially communicated, the cell culture chamber is surrounded, the micro-channel is communicated with the cell culture chamber by utilizing micro-column distribution, a circulating tumor culture medium is used as a circulating culture medium in a channel, and a tumor dormancy ecological bit stream is led to a perivascular ecological bit stream.
Description
Technical Field
The application relates to the technical field of microfluidics, in particular to an organ chip, an organ chip and application for constructing a malignant tumor bone metastasis ecological niche based on a microfluidic technology.
Background
Cancer is still one of the leading causes of human death worldwide, with about one-sixth of the deaths being caused by cancer. Reconstructing a realistic model of a microtumor niche has long been a great challenge facing the scientific community. Internal heterogeneity (i.e., cancer cells, mesenchymal cells, immune cells, vascular cells), other compounds (cytokines and growth factors) and the three-dimensionality of the tumor affect our understanding of the tumor niche and prevent our related replication in vitro.
Currently, less than 10% of clinically tested anticancer drugs are on the market, and most anticancer drugs end up failing in the final stage of clinical development, even though they have achieved positive results in preclinical trials. These facts indicate that the current model is unable to reproduce the tumor niche in vivo. They lack the necessary features required to mimic the complex cellular environment in vivo, such as cell-to-cell and cell-to-extracellular matrix interactions (differentiation, proliferation, morphology, gene expression, and other functions). On the one hand, organoids are highly variable in shape and size, and captured cells are difficult to quantify and visualize using microscopic techniques. On the other hand, the model lacks the signals and fluids that are commonly contacted in human tissue cells. The organ model on the microfluidic chip can better simulate the microstructure, dynamic mechanical property and biochemical function of the whole living organ and solid tumor, and can continuously provide nutrient substances and pharmaceutical compounds for the whole living organ and solid tumor, and the chip organ equipment currently used for the microfluidic chip covers a plurality of branches in the cancer field. Besides simulating tumor microenvironment and its characteristic region, it also reproduces tumor cell migration and invasion, metastasis model, vascularization and extravasation, tumor microenvironment reconstruction, immunooncology study, drug screening, etc.
The inventors have found in research that bone metastasis has become a persistent clinical problem, a significant cause of death in thousands of cancer patients. At present, most of anti-tumor bone metastasis treatments are palliative or supportive treatments, and only can delay the progress of the treatments, and radical measures are not yet available. Therefore, research on the generation mechanism of bone metastasis of lung cancer is important, wherein the bone microenvironment of metastasis is a research hot spot and difficulty, and may be a key breakthrough point for searching a method for effectively inhibiting and treating bone metastasis.
Disclosure of Invention
The application provides an organ chip for constructing a malignant tumor bone metastasis ecological niche based on a microfluidic technology, the organ chip and application thereof, and aims to solve the technical problem that effective inhibition and treatment of bone metastasis methods are required to be found in the prior art.
The application provides an organ chip for constructing a malignant tumor bone metastasis ecological niche, which comprises a tumor dormancy ecological niche, a perivascular ecological niche and a malignant circulation ecological niche, wherein:
the tumor dormancy ecological niche is used for wrapping bone resident cells and tumor cells;
the perivascular niche adopts an H-shaped vascular endothelial cell implantation channel for simulating a vascular system and the perivascular niche,
the malignant circulation niche is used for wrapping osteoclast and tumor cells in hydrogel injected into a chip culture cavity,
the H-shaped design comprises a micro-channel, a cell differentiation chamber for adjusting bone environment, and a chip culture cavity located in a malignant circulation ecological niche, wherein the micro-channel at least comprises a first micro-channel, a second micro-channel, a third micro-channel and a liquid outlet micro-channel, the first micro-channel, the second micro-channel, the third micro-channel and the liquid outlet micro-channel are sequentially communicated, a circulating tumor culture medium is used as a circulating culture medium in a channel, the tumor dormancy ecological bit stream flows to the niche around a blood vessel from the tumor dormancy ecological bit stream, and the liquid outlet micro-channel is connected after the first micro-channel is communicated with the cell differentiation chamber and the chip culture cavity.
The early stage of bone metastasis of malignant tumor has ecological niche reconstruction of primary tumor to bone metastasis, and relates to dormancy and reactivation of metastatic tumor, including tumor dormancy ecological niche, perivascular ecological niche and malignant circulation ecological niche. The organ chip simulates an H-shaped vascular structure of a bone at a transfer part, adopts an H-shaped runner design, and implants vascular endothelial cells on the peripheral wall of the channel for simulating a vascular system and a perivascular ecological niche. The H-shaped design comprises a micro-channel and a cell culture chamber, wherein the micro-channel at least comprises a first micro-channel, a second micro-channel, a third micro-channel and a liquid outlet micro-channel, the first micro-channel, the second micro-channel, the third micro-channel and the liquid outlet micro-channel are sequentially communicated, the cell culture chamber is surrounded, the micro-channel is communicated with the cell culture chamber by utilizing micro-column distribution, a circulating tumor culture medium is used as a circulating culture medium in a channel, and a tumor dormancy ecological bit stream is led to a perivascular ecological bit stream. The method comprises the steps of taking hydrogel as a three-dimensional culture system for cell culture, constructing a tumor dormancy ecological niche by combining a 3D printing Hydroxyapatite (HAP) bracket planted by mesenchymal stem cells, injecting loaded cell hydrogel into a cell culture chamber, observing cell differentiation of a bone microenvironment in the perivascular ecological niche in real time, and monitoring a reactivation process of tumors in the malignant circulation ecological niche by taking tumor invasive pseudopodia as an index. The chip platform is expected to become a reliable in-vitro platform for promoting research on bone metastasis and evaluation of metastasis risk of tumors by integrating key niches of bone microenvironment before tumor metastasis and reproducing cell activities of bone metastasis of malignant tumors in vitro.
Drawings
FIG. 1 is a schematic diagram of an organ-chip for constructing a malignant tumor bone metastasis niche according to the present application;
FIG. 2A is a chip channel design inspired by a limb bone H-shaped vessel; FIG. 2B is a schematic illustration of a limb bone H-shaped vessel; FIG. 2C is a streamed CAD image; FIG. 2D is a macroscopic view of the device injecting violet dye showing the distribution of the medium in the channels of the limb chip; fig. 2F is a chip channel design.
FIG. 3A is a schematic diagram of a chip channel inspired by H-shaped blood vessels of a vertebrate bone, and FIG. 3B is a schematic diagram of H-shaped blood vessels in a vertebrate bone; FIG. 3C is a CAD image with flow direction in a vertebrate chip; FIG. 3D is a macroscopic view of the device injecting violet dye showing the distribution of the medium in the channel; fig. 3F is a chip channel design.
FIG. 4 is a flow communication and cell viability of BOCs. (A) A fluid simulation and modeling diagram, a diagram (B) a nutrition traffic diagram between a channel and a hydrogel cavity captured by a time fluorescence microscope at a set time point; FIGS. 4 c-4 d are graphs showing staining by live/dead analysis after loading RAW264.7 cells into hydrogel injection for 7 and 14 days, respectively. (ii) In the drawing, the scale bar is (i) 100 μm, and (ii) 50 μm;
FIG. 5 is a graph of PMN simulated bone plateau cell distribution. Fig. 5 (a) implants MSCs on 3D printed (3 DP) scaffolds. Cytoskeleton was stained with Alexa Fluor 647 phalloidin (red) and nuclei were stained with DAPI (blue). FIG. 5 (B) Lung tumor cells (green) expressing green fluorescent protein A549 (GFP-A549) were loaded into GelMA hydrogels and injected into the msc seed scaffolds. FIG. 5 (C) schematic diagram of perivascular niche of chip platform. FIG. 5 (D) is a representative image of the perivascular niche of the chip platform;
FIG. 6 is a graph showing communication of tumor-osteoclasts at BOC. FIG. 6 (A) is a representative 3D view of intrusion formation in raw264.7 loaded GelMA hydrogels with or without CM. Scale = 40 μm. FIG. 6 (B) is a representative contact protein immunofluorescence image (i) and quantitative analysis of contact protein (ii) to assess the formation of a raw264.7 loaded GelMA hydrogel with or without CM in the chip cavity. FIG. 6 (C) RTqPCR results for invasive gene expression Tks5 (i) and cotacn (ii) when CM or RankL treated A549 cells (average S.E.M, <0.05, n=3). Fig. 6 (D) schematic of a long-term (7 days) co-culture model of H-type structure in BOC, where (i) represents a pooled cotacn fluorescence image of a549 cells under different co-culture conditions, (ii) quantitative results (average s.e.m, <0.05, n=6 per group). Scale = 40 μm. A549, control group; a549-RAW a549 and RAW264.7 separation chamber cells.
Detailed Description
The present application will be described in detail below with reference to the accompanying drawings.
Lung cancer is one of the most common malignant tumors in clinic, has a tendency to increase with years, has become one of the main causes of death of cancer, and is the most common in non-small cell lung cancer, with a survival rate of about 15% in 5 years. With the wide development of therapeutic intervention means of lung cancer including targeted therapy and immunotherapy, the overall life cycle of patients is prolonged, and the life quality is improved. However, advanced lung cancer is susceptible to metastasis, and it is counted that 30% -40% of lung cancer patients undergo bone metastasis. Bone metastasis of lung cancer is frequently seen as osteolytic, often accompanied by bone related events (skeletal related events, SRES) such as pain, hypercalcemia, pathological fractures, spinal cord compression, and even paraplegia, cancer cachexia. Bone metastasis has become a persistent clinical problem, a significant cause of death in thousands of cancer patients. At present, most of anti-tumor bone metastasis treatments are palliative or supportive treatments, and only can delay the progress of the treatments, and radical measures are not yet available. Therefore, research on the generation mechanism of bone metastasis of lung cancer is important, wherein the bone microenvironment of metastasis is a research hot spot and difficulty, and may be a key breakthrough point for searching a method for effectively inhibiting and treating bone metastasis.
Paget's "seed and soil" is one of the mainstream theories of tumor metastasis, and the microenvironment of the metastatic sites has an important impact on the success of tumor metastasis. Pre-metastatic niches (PMNs) refer to primary tumors that release tumor secretion factors and microvesicles to recruit bone marrow-derived cells in secondary organs to create a microenvironment suitable for tumor metastasis growth proliferation, providing a Pre-stage preparation for metastasis of primary foci. PMN formation indicates targeting of tumor metastasis and provides fertile "soil" for pre-transferred "seeds". The establishment of a strategy for preventing metastasis and early diagnosis and early treatment is a primary task for improving prognosis of lung cancer patients, so that the proposal of PMN is beneficial to early prevention and treatment of tumor metastasis. With the deep research on PMN formation, the mechanism of tumor metastasis is elucidated to a greater extent, and more effective clinical treatment means are formulated, so that the method has important significance in reducing the metastasis rate and the death rate of tumor patients and improving the prognosis of the tumor patients. The current research on the mechanism of tumor bone metastasis PMN is mainly focused on the promotion of PMN formation by vesicles or factors secreted by tumor cells, and is an exploration of the upstream cause of PMN formation, and the research on the mechanism of PMN formation is not yet traced back from downstream factors (such as the microenvironment of the target metastasis site).
Abnormal metabolism is an important marker of tumor metastasis. During the course of tumor metastasis, specific metabolic pathways are re-edited to support their transition through the metastatic cascade, thereby forming secondary tumors in distant organs. And in bone PMN formation, the particular microenvironment of the bone plays an important role. Physical factors in the bone marrow microenvironment such as hypoxia, acidic pH and high calcium ion concentration environments all promote tumor cell proliferation. The red bone marrow area has high blood flow, which is beneficial to the metastasis and growth of tumor cells. Bones act as important endocrine organs and regulate the general state. Bone microenvironment metabolism includes bone metabolism, namely continuous and circulating interaction of osteoblasts and osteoclasts to maintain dynamic balance of osteolysis and osteogenesis, and various factors released in the process are beneficial to the field planting and proliferation of tumor cells; on the other hand, the metabolism of stromal cells such as fibroblasts, endothelial cells, adipocytes, immune cells and the like can promote bone metastasis of tumor cells, and the two together determine the final stage of bone metastasis. The interaction between the factors secreted by tumor cells and the specific bone microenvironment allows for adaptive changes in both metabolism to support tumor bone metastasis progression. The metabolites produced by cellular metabolism are downstream products of PMN formation and can be further explored by metabonomics.
The current study of PMNs is still in an early stage, and its key reason is: the detection means of PMNs are limited. PET-CT imaging is not sensitive to tumor diagnosis of less than 1cm in diameter, and early detection of PMNs prior to radiological evidence metastasis remains a challenge; imaging techniques with higher resolution and capable of detecting structural changes (e.g., tissue density) in PMNs are being developed for use in the lack of clinical blood molecular markers; the research means is lacking, the microenvironment with complex tumor bone metastasis is difficult to reproduce in vitro, and the difference between the in vitro 2D culture effect and the real in vivo situation is too large; the animal body cannot be observed in real time, is expensive, has ethical problems and the like, so that a proper in vitro model is necessary for the study of the PMN formation mechanism. Organ-chip, also called micro-physiological system (Microphysiological system, MPS), simulates in vivo environment and organ function by culturing human cells in an in vitro chip, reconstructing the microstructure functional units of specific human organs and tissue-tissue interfaces, and giving dynamic mechanical and biochemical stimuli. For the above application scenario, my applicant invented an organ chip for constructing a malignant tumor bone metastasis ecological niche, and it should be noted that the organ chip is not only aimed at bone metastasis of lung cancer, please refer to fig. 1, which is a schematic diagram of the organ chip for constructing a malignant tumor bone metastasis ecological niche according to the present application. It includes tumor dormancy niche, perivascular niche and vicious circle niche, wherein:
the tumor dormancy ecological niche is used for wrapping bone resident cells and tumor cells;
the perivascular niche adopts an H-shaped vascular endothelial cell implantation channel for simulating a vascular system and the perivascular niche,
the malignant circulation niche is used for wrapping osteoclast and tumor cells in hydrogel injected into a chip culture cavity,
the H-shaped design comprises a micro-channel, a cell differentiation chamber for adjusting bone environment, and a chip culture cavity located in a malignant circulation ecological niche, wherein the micro-channel at least comprises a first micro-channel, a second micro-channel, a third micro-channel and a liquid outlet micro-channel, the first micro-channel, the second micro-channel, the third micro-channel and the liquid outlet micro-channel are sequentially communicated, a circulating tumor culture medium is used as a circulating culture medium in a channel, the tumor dormancy ecological bit stream flows to the ecological niche around a blood vessel from the tumor dormancy ecological bit stream, and the liquid outlet micro-channel is connected after the first micro-channel is communicated with the cell differentiation chamber and is communicated with the chip culture cavity through the third micro-channel.
The tumor dormancy niche photo-crosslinked methacrylic acid gelatin (GelMA) is used as a three-dimensional culture hydrogel, and wraps bone resident cells and tumor cells under blue light irradiation. The resting niche was reproduced using a Mesenchymal Stem Cell (MSC) implanted 3d printed Hydroxyapatite (HAP) chip scaffold. 3D printing technology is an effective way to construct tissue biological scaffolds. Compared with other 3D culture models, the three-dimensional culture model can accurately arrange cells and extracellular matrixes, control local tissue microenvironment, further construct in-vivo tissue-like forms and mechanical environments, and simulate in-vivo cell-cell interaction and cell-extracellular matrix interaction. In addition, the porosity inside the stent can be precisely controlled by blending materials and cells in advance and regularly arranging the materials and the cells after extrusion, so that oxygen and nutrient substance transmission and metabolic waste exchange are facilitated, and the survival of cells inside the stent is further maintained. In recent years, the construction of a 3D biological printing active scaffold with biological hydrogel loaded stem cells as printing ink has high potential in the tissue regeneration and repair directions. It mainly comprises three organic components: functional cells, biological scaffold material, and loaded bioactive components. Wherein the choice of the biological scaffold material and its supported bioactive components is the core of the technology. The micro-tissue construction can be better realized by means of a high-precision processing technology special for a 3D biological printing technology, so that microscopic components and structures of the tissue can be better simulated. In this example, a Mesenchymal Stem Cell (MSC) implanted 3d printed Hydroxyapatite (HAP) chip scaffold was used to reproduce the resting niche using the techniques described above.
The most important technical innovation of the application is as follows: the cell differentiation chamber is used for culturing the hydrogel chamber, and the chip culture chamber is used for inducing the formation of a tumor dormancy microenvironment. The H-shaped design comprises the following steps: the method comprises a micro-channel, a cell differentiation chamber for regulating bone environment, and a chip culture cavity located in a malignant circulation ecological niche, wherein the micro-channel at least comprises a first micro-channel, a second micro-channel, a third micro-channel and a liquid outlet micro-channel, the first micro-channel, the second micro-channel, the third micro-channel and the liquid outlet micro-channel are sequentially communicated, a circulating tumor culture medium is used as a circulating culture medium in a channel, the tumor dormancy ecological bit stream is sent to the ecological niche around a blood vessel from the tumor dormancy ecological bit stream, and the liquid outlet micro-channel is communicated with the cell differentiation chamber by the first micro-channel and the chip culture cavity through the third micro-channel. In the malignant circulation niche (namely a chip culture cavity), osteoclast and tumor cells are wrapped in hydrogel injected into the chip cavity, and a tumor dormancy microenvironment is formed by induction. The ring tumor medium (CM) served as a circulating medium in the channel, connecting the primary tumor and bone metastases.
The cell differentiation chamber/chip culture chamber is surrounded by a plurality of triangular columns to form a chamber, a circulation space is arranged between the triangular columns, the triangular columns and the triangular columns are used for separating micro-channels from the cell differentiation chamber/chip culture chamber, and circulating culture medium can flow into the chamber conveniently through the circulation space and surface tension is generated in space to effectively prevent the circulating culture medium from flowing out of the chamber. The triangular prism is columnar, the cylindrical surface of one corner is facing the outside of the chamber, the other two corners are arranged towards the inside direction of the chamber, and the circulating culture medium circulates towards the inside of the chamber along the cylindrical surface directions of the other two corners. The triangular column is designed to separate the channel and the chamber, generate surface tension, and effectively prevent the outflow of hydrogel solution, similar to the functional design of a one-way valve. The independence of the hydrogel is cultivated in the cell differentiation chamber, A549 tumor cells are filled into GelMA hydrogel, and the bone seed HAP bracket is combined to induce the tumor dormancy microenvironment.
The side of the cell differentiation chamber corresponding to the first micro flow channel is also provided with a non-circulating micro flow channel, and the arrangement is mainly used for enabling the cell differentiation chamber to be fully exchanged with the channel, exchanging the culture hydrogel into the cell differentiation chamber and exchanging the waste liquid. And, can only flow to the second little through-flow through first little through-flow, guarantee the unidirectional nature of circulation.
Similarly, the provision of the second micro flow channel also provides the possibility of exchanging between the cell differentiation chamber and the chip culture chamber. The side of the chip culture chamber corresponding to the third micro flow channel is also provided with a micro flow channel which does not circulate. Also, the space of the exchange channel is increased, and sufficient exchange can be obtained.
For this purpose, the platform may also be designed with a monitoring unit for monitoring the formation of pseudopodia to verify the reactivation of metastatic tumor cells.
For example, peristaltic pumps are respectively connected with two liquid outlets or one liquid outlet, and circulation perfusion is carried out at a certain speed (such as a flow rate of 5 ul/min) to simulate blood flow in a body; so that the cell differentiation chamber flows into the circulating culture medium, the osteoclast and the tumor cells are wrapped in the hydrogel injected into the chip cavity, and the tumor dormancy microenvironment is induced to simulate the microenvironment of the bone tissue in the body.
First example
Please refer to fig. 2A-2F, which are exemplary diagrams of an organ-chip. Including the chip passageway that receives limb bone H type blood vessel inspires, at least first inlet and a plurality of H type design, H type design includes the microchannel, is used for the cell differentiation cavity of bone environment regulation, is located the chip culture chamber of vicious circulation ecological niche, the microchannel includes first microchannel, second microchannel and third microchannel and play liquid microchannel at least to, first microchannel, second microchannel, third microchannel and play liquid microchannel communicate in proper order, and circulation tumour culture medium is as the circulation culture medium in the passageway, connects a plurality of limb bone H type blood vessels respectively from first inlet, utilizes first microchannel and cell differentiation cavity intercommunication, connects out liquid microchannel after the third microchannel and chip culture chamber intercommunication.
The chip culture cavity further comprises the following components: a549 tumor cells were loaded into GelMA hydrogels and combined with the msc seed HAP scaffold to form an induced tumor dormancy microenvironment. The first liquid inlet is provided with a printing assembly HAP bracket. The chip can further comprise second liquid inlets which are respectively connected with a plurality of H-shaped designs of the limb bones, and the second liquid inlets are provided with photo-crosslinking methacrylic acid gelatin GelM which is used as the three-dimensional culture hydrogel.
Preparation and characterization of a sub-transfer ecological niche simulation BOC platform. Fig. 2 is a chip channel design inspired by limb bone H-type vessels. FIG. 2B is a schematic illustration of a limb bone H-shaped vessel; FIG. 2C is a streamed CAD image; fig. 2D is a macroscopic view of the device injecting violet dye showing the distribution of the medium in the channels of the extremity chip. Fig. 2F is a chip channel design.
Second example
Referring to fig. 3A-3D, which are schematic views of another example of an organ chip, the organ chip includes a chip channel inspired by a vertebrate bone H-shaped vessel, at least includes a first liquid inlet, a second liquid inlet and a plurality of vertebrate bone H-shaped designs, the vertebrate bone H-shaped designs include a micro-channel, a cell differentiation chamber for adjusting bone environment, a chip culture chamber located in a malignant circulation ecological niche, the micro-channel includes at least a first micro-channel, a second micro-channel, a third micro-channel and a liquid outlet micro-channel, the first micro-channel, the second micro-channel, the third micro-channel and the liquid outlet micro-channel are sequentially communicated, a circulating tumor culture medium flows to each limb bone H-shaped design from two ends of the first liquid inlet and the second liquid inlet, and then is connected with the liquid outlet micro-channel after being communicated with the chip culture chamber by the first micro-channel and the third micro-channel arranged by each H-shaped design. Fig. 3A shows a chip channel inspired by a vertebrate bone H-vessel. FIG. 3B is a schematic view of an H-shaped vessel in a vertebrate bone; FIG. 3C is a CAD image with flow direction in a vertebrate chip; fig. 3D is a macroscopic view of the device injecting violet dye showing the distribution of the medium in the channel. Fig. 3F is a chip channel design. Dye flow in the H-type structure to demonstrate the independence of (i) fluid channels and (ii) co-culture hydrogel chambers. The triangular column is designed to separate the channel and the chamber, so that surface tension is generated, and the outflow of the hydrogel solution can be effectively prevented. (D) schematic drawing of the 3D printing bone tumor scaffold. A549 tumor cells are filled into GelMA hydrogel and combined with the msc seed HAP bracket to induce a tumor dormancy microenvironment. Printing assembled HAP scaffolds with or without GelMA hydrogel (red) on both types of chip platforms.
FIG. 4 is a flow communication and cell viability of BOCs. (A) fluid simulation and modeling. Velocity and pressure profiles before (i, ii) and after (iii, iv) hydrogel injection into the chamber. The red arrows indicate the flow direction. The change in color from red to blue indicates a decrease in speed or pressure. (B) The time fluorescence microscope captured the nutrient communication between the channel and the hydrogel cavity at the set time point. FITC-dextran (70 kDa) (time new roman) diffuses into the channel (ii) of GelMA hydrogel (i) at 30 s for 15, 30, 45, 60 minutes. Scale bar = 0.5 mm. (c-d) RAW264.7 cells were loaded into the hydrogel for 7 and 14 days after injection, as indicated by live/dead analytical staining. (ii) In the drawing, the scale of (i) the scale is 100 μm and (ii) the scale is 50. Mu.m
FIG. 5 is a graph of PMN simulated bone plateau cell distribution. (A) implantation of MSCs on 3D printing (3 DP) scaffolds. Cytoskeleton was stained with Alexa Fluor 647 phalloidin (red) and nuclei were stained with DAPI (blue). (B) Lung tumor cells (green) expressing green fluorescent protein A549 (GFP-A549) were loaded into GelMA hydrogels and injected into the msc seed scaffolds. And (C) a schematic diagram of the perivascular niche of the chip platform. (D) Representative images of perivascular niches of the chip platform, where green cells are a549 cells loaded in hydrogels and red cells are HUVECs spread around the channel. (E-F) detailed distribution of huvec cells over the depth of the chip channels. Scale = 100 μm. .
FIG. 6 is a graph showing communication of tumor-osteoclasts at BOC. (A) Representative 3D view of intrusion formation in raw264.7 loaded GelMA hydrogels with or without CM. Scale = 40 μm. (B) Representative contact protein immunofluorescence images (i) and quantitative analysis of contact proteins (ii) were performed to assess the formation of gel-ma hydrogels loaded with raw264.7 with or without CM intrusion into the chip cavity. A549 cells were marked green, surface was marked red, and nuclei were marked blue (average s.e.m., <0.05, n=6 per group). Scale = 40 μm. (C) RTqPCR results (average s.e.m, <0.05, n=3) of invasive gene expression Tks5 (i) and cotacn (ii) when CM or RankL treated a549 cells. (D) Schematic of a long-term (7 days) co-culture model of H-type structure in BOC, where (i) represents a pooled cotacn fluorescence image of a549 cells under different co-culture conditions, (ii) quantitative results (mean s.e.m, <0.05, n=6 per group). Scale = 40 μm. A549, control group; a549-RAW: a549 and RAW264.7 separation chamber cells; a549/RAW a549 and RAW264.7 units are in the same chamber.
An organ chip application, which is applied to an in vitro tool of pmn formation mode in lung cancer bone metastasis, wherein the in vitro tool is any one of the organ chips.
The above description is only a preferred embodiment of the present application, and the protection scope of the present application is not limited to the above examples, and all technical solutions belonging to the concept of the present application belong to the protection scope of the present application. It should be noted that modifications and adaptations to the present application may occur to one skilled in the art without departing from the principles of the present application and are intended to be within the scope of the present application.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
Claims (11)
1. An organ chip for constructing a malignant tumor bone metastasis niche, comprising a tumor dormancy niche, a perivascular niche, and a malignant circulation niche, wherein:
the tumor dormancy ecological niche is used for wrapping bone resident cells and tumor cells;
the perivascular niche adopts an H-shaped vascular endothelial cell implantation channel for simulating a vascular system and the perivascular niche,
the malignant circulation niche is used for wrapping osteoclast and tumor cells in hydrogel injected into a chip culture cavity,
the H-shaped design comprises a micro-channel, a cell differentiation chamber for adjusting bone environment, and a chip culture cavity located in a malignant circulation ecological niche, wherein the micro-channel at least comprises a first micro-channel, a second micro-channel, a third micro-channel and a liquid outlet micro-channel, the first micro-channel, the second micro-channel, the third micro-channel and the liquid outlet micro-channel are sequentially communicated, a circulating tumor culture medium is used as a circulating culture medium in a channel, the tumor dormancy ecological bit stream flows to the niche around a blood vessel from the tumor dormancy ecological bit stream, and the liquid outlet micro-channel is connected after the first micro-channel is communicated with the cell differentiation chamber and the chip culture cavity.
2. The organ chip according to claim 1, wherein the cell differentiation chamber/chip culture chamber is surrounded by a plurality of triangular columns, a circulation space is provided between the triangular columns, the triangular columns and the triangular columns are provided for separating the micro-channel from the cell differentiation chamber/chip culture chamber, and the circulation space is convenient for flowing the circulating culture medium into the chamber and generating surface tension in space to effectively prevent the circulating culture medium from flowing out of the chamber.
3. The organ-chip according to claim 2, wherein the triangular prism is cylindrical, the cylindrical surface of one corner faces the outside of the chamber, the other two corners face the inside of the chamber, and the circulating culture medium is circulated to the inside of the chamber along the cylindrical surfaces of the other two corners.
4. The organ-chip according to claim 1, wherein photo-crosslinked gelatin methacrylate GelMA is used as a three-dimensional culture hydrogel, encapsulating bone resident cells and tumor cells under blue light irradiation, reproducing dormant niches using a 3d printed hydroxyapatite HAP chip scaffold implanted with mesenchymal stem cells MSCs.
5. The organ-chip of claim 1, further comprising: the method comprises the steps of taking hydrogel as a three-dimensional culture system for cell culture, combining a 3D printing hydroxyapatite HAP bracket planted by mesenchymal stem cells to construct a tumor dormancy ecological niche, injecting loaded cell hydrogel into a cell culture chamber, observing cell differentiation of bone microenvironment in the perivascular ecological niche in real time, monitoring a reactivation process of tumors in a malignant circulation ecological niche by taking tumor invasive pseudopodia as an index, and reproducing cell activity of bone metastasis of malignant tumors in vitro by integrating a key ecological niche of the bone microenvironment before tumor metastasis.
6. The utility model provides an organ chip, includes the chip passageway that receives limb bone H type blood vessel inspires, its characterized in that, at least first inlet and a plurality of H type design, H type design includes the microchannel, be used for the cell differentiation cavity of bone environment regulation, be located the chip culture chamber of vicious circle ecological niche, the microchannel includes at least first microchannel, second microchannel and third microchannel and play liquid microchannel, and, first microchannel, second microchannel, third microchannel and play liquid microchannel communicate in proper order, circulation tumour culture medium is as the circulation culture medium in the passageway, connect a plurality of limb bone H type blood vessels respectively from first inlet, utilize first microchannel and cell differentiation cavity intercommunication, connect out the liquid microchannel after the third microchannel and chip culture chamber intercommunication.
7. The organ chip of claim 6, wherein the cell culture is performed by using hydrogel as a three-dimensional culture system, a tumor dormancy niche is constructed by combining a 3D-printed hydroxyapatite HAP scaffold planted by mesenchymal stem cells, the loaded cell hydrogel is injected into a cell culture chamber to observe cell differentiation of bone microenvironment in perivascular niches in real time, the reactivation process of tumors in the malignant circulation niche is monitored by using tumor invasive pseudopodia as an index, and the cell activity of bone metastasis of malignant tumors is reproduced in vitro by integrating the key niche of the bone microenvironment before tumor metastasis.
8. The organ-chip according to claim 6, wherein the first inlet is provided with a print assembly HAP scaffold.
9. The organ-chip according to claim 6, further comprising second liquid inlets, said second liquid inlets being respectively connected to a plurality of limb-bone-receiving H-shaped designs, said second liquid inlets being provided with photo-cross-linked gelatin methacrylate GelM to be used as a three-dimensional culture hydrogel.
10. The utility model provides an organ chip, includes the chip passageway that receives vertebrate skeleton H type blood vessel heuristic design, its characterized in that includes first inlet, second inlet and a plurality of design of receiving vertebrate skeleton H type at least, receive the design of vertebrate skeleton H type and include the microchannel, be used for the cell differentiation cavity of bone environment regulation, be located the chip culture chamber of vicious circle ecological niche, the microchannel includes at least first microchannel, second microchannel and third microchannel and play liquid microchannel, and first microchannel, second microchannel, third microchannel and play liquid microchannel communicate in proper order, circulation tumour culture medium is as the circulation culture medium in the passageway, flow to every and receive limb bone H type design from first inlet and second inlet both ends respectively, respectively utilize the first microchannel and the cell differentiation cavity intercommunication of every H type setting, connect the play liquid microchannel after third microchannel and chip culture chamber intercommunication.
11. Use of an organ-chip, characterized in that it is applied to an in vitro tool for the pmn formation mode in bone metastases of lung cancer, which comprises an organ-chip according to any one of claims 1 to 10.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310860325.6A CN116875459A (en) | 2023-07-13 | 2023-07-13 | Organ chip for constructing malignant tumor bone metastasis ecological niche, organ chip and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310860325.6A CN116875459A (en) | 2023-07-13 | 2023-07-13 | Organ chip for constructing malignant tumor bone metastasis ecological niche, organ chip and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116875459A true CN116875459A (en) | 2023-10-13 |
Family
ID=88258221
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310860325.6A Pending CN116875459A (en) | 2023-07-13 | 2023-07-13 | Organ chip for constructing malignant tumor bone metastasis ecological niche, organ chip and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116875459A (en) |
-
2023
- 2023-07-13 CN CN202310860325.6A patent/CN116875459A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11976294B2 (en) | Hypothermic 3D bioprinting of living tissues supported by perfusable vasculature | |
| CN102899249B (en) | Produce the apparatus and method of extracorporeal blood vessel | |
| JP6928598B2 (en) | Human and large mammalian lung bioreactors | |
| Elliott et al. | A review of three-dimensional in vitro tissue models for drug discovery and transport studies | |
| KR20180027553A (en) | Vascularized test tube perfusion device, manufacturing method and its application method | |
| KR20150063077A (en) | Multi-organ-chip with improved life time and homoeostasis | |
| Paggi et al. | Joint-on-chip platforms: entering a new era of in vitro models for arthritis | |
| Malik et al. | Critical considerations for the design of multi-organ microphysiological systems (MPS) | |
| RU171690U1 (en) | Microfluidic Chip for the Creation of Cellular Models of Mammalian Organs | |
| JP2017501745A (en) | Fluidic device and perfusion system for the reconstruction of complex biological tissue outside the body | |
| US20210108178A1 (en) | Systems and methods for multilane vasculature | |
| WO2019237061A1 (en) | A 3d bioprinted perfusable platform for high-throughput disease modeling and drug screening | |
| US9260686B2 (en) | Tubular bioreactor system for use in bone and cartilage tissue engineering | |
| De Lorenzi et al. | Engineering Mesoscopic 3D Tumor Models with a Self‐Organizing Vascularized Matrix | |
| Kim et al. | Bone-on-a-chip: biomimetic models based on microfluidic technologies for biomedical applications | |
| CN116875459A (en) | Organ chip for constructing malignant tumor bone metastasis ecological niche, organ chip and application | |
| Kim et al. | Multiscale engineered human skeletal muscles with perfusable vasculature and microvascular network recapitulating the fluid compartments | |
| CN115895895A (en) | Blood vessel series-connected multi-organ chip model and application method thereof | |
| Nguyen et al. | Translational strategy: humanized mini-organs | |
| Kolahi Azar et al. | The progressive trend of modeling and drug screening systems of breast cancer bone metastasis | |
| CN219490026U (en) | Three-dimensional dynamic culture colorectal tissue-like tumor chip | |
| Bauer-Rowe et al. | QS1: creeping fat adipocytes drive intestinal fibrosis through adipocyte-to-fibroblast conversion in a novel model of inflammatory bowel disease | |
| US11969727B2 (en) | Tumor microenvironment on chip | |
| CN116814421A (en) | Organ chip for multi-tissue co-culture and detection method for multi-tissue function regulation | |
| CN116814527A (en) | In-vitro tumor heart blood vessel model and method for detecting compound by using same |
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 |