CN108330099A - The culture of personalized liver cell and amplification method and its application - Google Patents

The culture of personalized liver cell and amplification method and its application Download PDF

Info

Publication number
CN108330099A
CN108330099A CN201710172028.7A CN201710172028A CN108330099A CN 108330099 A CN108330099 A CN 108330099A CN 201710172028 A CN201710172028 A CN 201710172028A CN 108330099 A CN108330099 A CN 108330099A
Authority
CN
China
Prior art keywords
culture
liver cell
liver
parenchymal cells
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201710172028.7A
Other languages
Chinese (zh)
Inventor
吴红平
周徐
金晓芳
曾敏
黄伟健
张洪丹
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Sailiwei Biotechnology Co Ltd
Original Assignee
Shanghai Sailiwei Biotechnology Co Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Shanghai Sailiwei Biotechnology Co Ltd filed Critical Shanghai Sailiwei Biotechnology Co Ltd
Priority to CN201710172028.7A priority Critical patent/CN108330099A/en
Publication of CN108330099A publication Critical patent/CN108330099A/en
Pending legal-status Critical Current

Links

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
    • C12N5/067—Hepatocytes
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/407—Liver; Hepatocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5067—Liver cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/106—Primate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/28—Materials or treatment for tissue regeneration for liver reconstruction
    • 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
    • C12N2501/105—Insulin-like growth factors [IGF]
    • 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
    • C12N2501/18—Liver cell growth factor (LCGF, Gly-His-Lys)
    • 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
    • C12N2510/00—Genetically modified 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
    • C12N2710/22022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Immunology (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Physics & Mathematics (AREA)
  • Virology (AREA)
  • Biophysics (AREA)
  • Urology & Nephrology (AREA)
  • Dermatology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Botany (AREA)
  • Environmental Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Hematology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Plant Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Animal Husbandry (AREA)
  • Pathology (AREA)

Abstract

The present invention relates to a kind of culture of personalized liver cell and amplification methods, which is characterized in that includes the following steps：Step 1 carries out genetic modification, to inhibit expression or the function of P53 genes to the hepatic parenchymal cells of acquisition；Step 2 will be cultivated and be expanded in the hepatic parenchymal cells merging hepatocyte growth culture medium for passing through genetic modification in step 1, obtains liver cell；Step 3, the liver cell that step 2 is obtained are placed in culture in hepatocyte differentiation culture medium and obtain hepatic parenchymal cells.The liver cell being prepared according to the above method, overcome in the past can not in vitro long-term cultivation and amplification hepatic parenchymal cells problem, provide unlimited cell origin for the application of liver cell.

Description

The culture of personalized liver cell and amplification method and its application
Technical field
The present invention relates to biotechnologies, and in particular to it is a kind of personalization liver cell culture and amplification method.
Background technology
Liver is the important vital organ based on metabolic function in animal body, regulates and controls different physiological roles, main to wrap Removing toxic substances is included, hepatic glycogen is stored, synthesize secreted protein and generate bile etc..Liver itself has powerful power of regeneration, Normal liver after 2/3rds partially hepatectomizeds, remaining mature hepatocytes can fast breeding, and completely replace and restore lack Lose the volume and function of liver.But suffering from various liver diseases, including hepatitis, hepatic sclerosis, liver cancer, liver metabolism disease and When liver failure, liver gradually loses power of regeneration, constantly declines so as to cause its physiological function, finally endangers the strong of people Health and life.
The patient that the whole world is influenced by hepatopathy is more than 500,000,000, once End-stage liver disease or acute hepatic failure are developed into, at present only One effective treatment means are to carry out liver transplant.But since liver donor continues in short supply and Postoperative Immunity system to allosome The development of the repulsion of organ, transfer operation receives serious restriction.The technology based on liver cell therapy receives extensively recently Concern, including hepatocyte transplantation, the reconstruction in vitro of liver organization and bioartificial liver etc., it is considered to be full organ transplant Comparatively ideal alternative.However, due to mature hepatocytes can not long-term in vitro culture and proliferation, this method continues to rely upon tax The donor liver offered, and the immunological rejection after transplanting cannot be changed.In addition, the liver cell of in vitro culture or extraordinary medicine Object liver metabolism model and hepatitis virus research model, are largely used by vast drugmaker and research institution.Therefore how Stable and a large amount of hepatocyte origin is obtained, current most urgent need is become.
In past many decades, researcher has carried out various trials, it is expected that it is peculiar to reappear liver cell in vitro Powerful proliferation potential.However so far, there is no report can in vitro long-term cultivation and be proliferated various animals normal hepatocytes it is real Cell plastid.In order to overcome the growth limitation of primary hepatocyte in vitro, researcher is by importing SV40 virus protein (large T antigens Or small T antigens) realize the immortalization (such as Fa2N-4 cell lines) of normal liver cell.More in addition come from the cell line of tumour It is established, and shows the form and function (such as HepaRG cell lines) of some mature hepatocytes.Although these cells can Infinitely culture and proliferation in vitro, but (including p450 is active for the exception for security reasons and on hepatocyte function It is heterogeneous), their practical application is extremely restricted, and is usually only used as drug metabolism and toxicity screening model, in some systems Medicine company is used (VallierL., HepswithPep on a small quantity: DirectReprogrammingintoHumanHepatocytes, CellStemCell, 14, March6,2014,267-269).
Recently, using stem cell or other cells come break up obtain liver cell be considered as the following liver cell it is important come Source.Induced multipotent stem cells (iPS) and pedigree reprogram the birth of technology even more so that establishing individuation liver cell, and final Realize that personalized medicine becomes possible.Have at present multiple reports can successfully by the embryonic stem cell of mammal (ES) and IPS cell directionals are divided into the cell with liver function.In addition, researcher is also successfully reprogrammed body cell by pedigree Direct transdifferentiation is liver cell, and proves that these cells can be proliferated and rebuild liver function in liver.But it is no matter thin by ES Born of the same parents, iPS cells or pedigree reprogramming differentiation obtain liver cell, are impossible to reach absolutely efficiency, and remaining is dry thin Born of the same parents are likely to form teratoma and adenoma after being injected into animal body, therefore have seriously affected the application of these cells.In addition, ES is thin Born of the same parents' materials are difficult and by the stringent limitation in ethics, and iPS cells and pedigree reprogramming all refer to multiple foreign genes and lead Enter, safety and hepatocyte function are similar to the liver cell of above-mentioned immortalization, therefore their practical application also had compared with the day of one's doom System.
Therefore, optimal hepatocyte origin or primary hepatocyte, if the long-term in vitro of primary hepatocyte can be realized Culture and proliferation, and keep the stability of normal liver cell science of heredity and function, will greatly push the mankind to the understanding of liver and The diagnosis and treatment of hepatopathy.
Liver cell is in the transplantation treatment of various hepatopathys, in the preparation for assisting liver function equipment, in drug toxicity Demand in screening and new drug development and in the fields such as hepatopathy and hepatitis research is huge.Since primary hepatocyte can not be in vitro Long-term cultivation simultaneously loses function quickly, and the medicament metabolism ability of primary hepatocyte shows significant inter-individual difference, therefore Endless functional personalized liver cell of supply will be greatly facilitated drug development and bioartificial liver and hepatocyte transplantation Final clinical application.
Technology disclosed in patent CN105296418A overcomes the problem that can not cultivate and expand in vitro in the past liver cell, Realize the long-term in vitro culture of individual specificity liver cell.The technology induces primary liver parenchyma thin by changing condition of culture Born of the same parents expand and are divided into again functional ripe hepatic parenchymal cells in vitro, but the liver parenchyma cultivated under the condition of culture The generation of cell secondary culture is relatively low, only 10-15 generations, the inefficiency of hepatocyte cultures and amplification, cannot meet extensive Application demand, cultivating the hepatic parenchymal cells of acquisition can degenerate after culture number generation, can not realize unlimited increasing.
Invention content
In view of the defects existing in the prior art, the purpose of the present invention is to provide a kind of cultures and expansion of personalized liver cell Increasing method realizes the infinite multiplication of ripe hepatic parenchymal cells in vitro.
Another object of the present invention is to provide the liver cells being prepared according to the above method.
Further aim of the present invention is the liver cell being prepared using the above method in personalized liver cell library Establish, compound and drug toxicology and pharmacological evaluation, hepatitis virus research and diagnosis and treatment, hepatocyte transplantation treatment, Prepare the application of preparation of bioartificial liver or humanization Chi-meric mice etc..
The first aspect of the present invention, there is provided a kind of cultures of personalized liver cell and amplification method, this method to include Following steps：
Step 1：Genetic modification is carried out to the hepatic parenchymal cells of acquisition, to inhibit expression or the function of P53 genes；
Step 2：It will be trained in the hepatic parenchymal cells merging hepatocyte growth culture medium for passing through genetic modification in step 1 It supports and expands, obtain liver cell；
Step 3：It is thin to be placed in the ripe liver of culture acquisition in hepatocyte differentiation culture medium for the liver cell that step 2 is obtained Born of the same parents..
Further, the method for carrying out genetic modification to the hepatic parenchymal cells described in step 1 is selected from channel genes, base Because of at least one of editor or RNA interference.
Further, the gene that the channel genes use is selected from the gene for inhibiting P53 expression or function, the inhibition P53 is expressed or the gene of function is selected from SV40 virus large T antigens gene, HPV viruse E6E7 genes or ubiquitin ligase gene；Institute It states ubiquitin ligase gene and is selected from least one of Mdm2, Hdm2, COP1, Pirh2 or ARF-BP1 gene, the gene Editor is selected from least one of CRISPR/Cas9, TALEN or ZFN method.
Further, the method for the channel genes is selected from the gene expression or non-virus carrier mediation that viral vectors mediates Gene expression.
Further, the viral vectors is selected from slow virus carrier, retroviral vector or gland relevant viral vector At least one of.
Further, the non-virus carrier in carrier for expression of eukaryon or transposons expression vector at least one Kind.
Further, the hepatic parenchymal cells are mammal hepatic parenchymal cells, the mammal hepatic parenchymal cells It is thin selected from mouse hepatocytes, rat hepatic parenchymal cells, rhesus macaque hepatic parenchymal cells, orangutan hepatic parenchymal cells or people's liver parenchyma It is one or more in born of the same parents.
Further, the hepatic parenchymal cells by genetic modification are attached on culture support, and it is thin to add liver What born of the same parents' proliferated culture medium was cultivated and was proliferated, the culture support is wrapped in advance using collagen or extracellular matrix protein Quilt；The extracellular matrix protein preferably is selected from Matrigel matrigels.The culture support refers to culture dish or culture plate.
Further, the hepatocyte growth culture medium includes basal medium, and the basal medium is selected from At least one in MEM, DMEM, BME, DMEM/F12, RPMI1640, William E, Neurobasal or Fischers culture medium Kind；
Preferably, the hepatocyte growth culture medium further includes nutrition, and the nutrition includes serum, serum In substitute, growth factor, the agonist of Wnt signal paths, Rho related protein kinases enzyme inhibitor, ALK5 kinase inhibitors It is at least one.
Further, the serum substitute is selected from KnockOutTM serum substitutes, Insulin-Transferrin-Asia At least one of sodium selenate mix supplement liquid, N2 nutritional supplements, B27 nutritional supplements.
Further, the growth factor is selected from epidermal growth factor, hFGF-2, liver cell At least one of growth factor, platelet cell growth factor or insulin-like growth factor.
Further, the Wnt pathway agonists be selected from CHIRP98014, TWS119, CHIRP99021, Wnt3a or At least one of R-Rspondin.
Further, the Rho related protein kinase enzyme inhibitors be selected from thiazovivin, Y-27632 or At least one of Blebbistatin.
Further, the ALK5 kinase inhibitors are selected from least one of SB431542, A83-01, RepSox.
Preferably, the nutrition includes 1-100ng/ml human hepatocyte growth factors, and 0.1-10 μM of Wnt signal is logical Road agonist CHIR99021,0.1-100 μM of Rho related protein kinase enzyme inhibitors Y-27632, volumn concentration 0.1-10% Insulin-Transferrin-sodium selenite mix supplement liquid basal cell culture medium or 0.1-100 μM of ALK5 kinase inhibition Agent A83-01.Wherein, the content of human hepatocyte growth factor is preferably 20ng/ml；Wnt signal path agonists CHIR99021 Content be preferably 3 μM, the content of Rho related protein kinase enzyme inhibitors Y-27632 is preferably 10 μM, ALK5 kinase inhibitors The content of A83-01 is preferably 1M, and the content of Insulin-Transferrin-sodium selenite mix supplement liquid is preferably 1%.
Further, the hepatocyte differentiation culture medium is made of basal medium and additive, the basal cell Culture medium is selected from MEM, DMEM, BME, DMEM/F12, RPMI1640, William E, Neurobasal or Fischers culture medium At least one of；The additive include serum substitute, hepatocyte growth factor, dexamethasone, people tumour inhibitor-M or At least one of ALK5 kinase inhibitors.
Further, the serum substitute is selected from KnockOutTM serum substitutes, Insulin-Transferrin-Asia It is one or more in sodium selenate mix supplement liquid, N2 nutritional supplements, B27 nutritional supplements.
Preferably, the basal medium is WilliamsE or DMEM/F12 culture mediums；The additive includes The serum or serum substitute of 5%-10% percents by volume, the human hepatocyte growth factor of 1-100ng/ml, 0.01-100 μM Dexamethasone, 1-100 μM of people's tumour inhibitor-M, 1-100 μM of ALK5 kinase inhibitors SB431542.Wherein, serum substitute The Insulin-Transferrin of preferably 5% percent by volume-sodium selenite mix supplement liquid；The human hepatocyte growth factor Content be preferably 20ng/ml；The content of the dexamethasone is preferably 1-10 μM；The content of people's tumour inhibitor-M is preferably 10-50μM；The content of the ALK5 kinase inhibitors SB431542 is preferably 10 μM.
The pH of above-mentioned hepatocyte growth culture medium and hepatocyte differentiation culture medium can be the normal of culture mammalian cell Advise pH, such as pH7.2-7.6.
Further, the step 2 further includes：Passage training is carried out to the liver cell after cultivating and be proliferated 7-10 days It supports and expands.After the hepatic parenchymal cells are adherent, cellular morphology changed at 3-5 days, cell elongate and may occur in which it is multiple to The protrusion of outer stretching, extension, nucleus volume become larger；There is typical epithelial cell form and start fast breeding in 5-7 days cells, carefully Born of the same parents' doubling time is 16-20 hours；Cell can be passed on and be continued to multiply within 7-10 days, cell can in vitro continuous passage 30 times with On.
Further, the liver cell secondary culture and succeeding generations include：It is added by 50 μ l/cm2 after sucking culture medium Cell dissociation buffer digests 5-10 minutes, makes the liver cell is completely agglomerating from culture support to fall off, is added and contains serum free culture system Base weight is hanged and is centrifuged, and centrifugal condition is to be centrifuged 5 minutes at 150G, and the liver cell passage ratio is 1:2-1:4.
The liver cell can freeze and recover repeatedly, freeze identical with ordinary cells with method for resuscitation.
The hepatic parenchymal cells are purchased from Invitrogen companies, also can voluntarily be prepared by literature method (MaurelP., Hepatocytes-MethodsandProtocols, METHODSINMOLECULARBIOLOGY, ISSN1064-3745).
Further, the present invention includes the marker identification for the liver cell that step 3 obtains.
Gene expression：Mature hepatocytes characteristic transcription factor such as HNF4 α, HNF6, Prox1；For example white egg of functioning gene (ALB) in vain, alpha-1 antitrypsin (AAT), G-6-Pase (G6P)；Drug metabolism I phases enzyme such as CYP1A2, CYP2C9, CYP3A4；Drug metabolism II phases enzyme such as MGST1 and UGT1A1；Drug metabolism III phases transport son such as NTCP, MRP2 and OATP1B3.
Protein expression：Flow cytometry albumin (ALB), asialoglycoprotein receptor (ASGPR).Immunofluorescence is examined Survey albumin (ALB), alpha-1 antitrypsin (AAT), cadherin E (E-cadherin), HNF4 α etc..
Protein secretion：Enzyme-linked immunosorbent assay detects the level for being secreted into albumin in cells and supernatant.
Biological function：Periodic acid avenges husband (PAS) and dyes detection glycogen storage；BODIPY dyeing detection lipid synthesis；Urea It generates and is detected using kit.
Detoxicating activity：P450-GloTM fluorescein enzyme process detects CYP3A4, the enzyme activity level of CYP1A2, CYP2C9.
Further, the application the present invention provides the liver cell in establishing personalized liver cell library.
Further, the present invention provides the liver cells in compound and the toxicology and pharmacological evaluation of drug Application.
Further, the answering in preparing diagnosis or treatment hepatitis virus medicament the present invention provides the liver cell With.
Further, the application the present invention provides the liver cell in preparing bioartificial liver's material.
Further, the present invention provides the liver cells moves the application in growing in liver cell.
Further, the application the present invention provides the liver cell in the preparation of humanization Chi-meric mice.
Term：
As used herein, unless otherwise stated, " hepatic parenchymal cells " refer to coming from mammal such as human liver tissue Fresh separated or the liver parenchymal cell frozen.
As used herein, " mammal " is Chordata (Chordata) Vertebrate (Vertebrata) animal of Mammalia (Mammalia).Mammal of the present invention includes people, also includes inhuman lactation Animal.The non-human mammal is, for example, mouse, rat, rabbit, dog, rabbit, ape and monkey, pig, ox, sheep, horse etc..No matter right and wrong People mammal or people, in the forming of genome, the side such as development, metabolic way, anatomical organ, disease incidence mechanism of individual Face is all very close.During evolution, some crucial cell function or regulatory pathway right and wrong between different species Often conservative.For example the signal path of cell Proliferation, apoptosis is almost the same in mammals.The aging approach of cell is also one A very conservative regulatory mechanism.In certain embodiments of the present invention, it is compared with the mankind using mouse as model organism, it No matter all connect very much with the mankind in development, metabolic way, anatomical organ, the disease incidence mechanism etc. of the forming of genome, individual Closely；Therefore, the present invention enumerate some be suitable for people the case where can unambiguously be suitable for non-human mammal.
As used herein, " extracellular matrix protein " refers to the extracellular basement membrane proteins of multi-cellular structure, mainly The effect for playing structural support and function point analysis, includes but are not limited to Collagen, Fibronectin, Vitronectin, Vimentin, Gelatin, Matrigel (BD), Geltrix (Invitrogeninc), StemAdhere (StemcellTechnology) etc..Illustrate by taking Matrigel as an example, Matrigel is by BD companies from rich in extracellular matrix Isolate BDMatrigel basement membrane matrixs in the EHS mouse tumors of protein mixture, main component is by laminin, and IV Collagen type, nestin, the compositions such as heparin sulfate glycoprotein also include growth factor and matrix metalloproteinase etc.. At ambient temperature, polymerization forms the three dimensional matrix with biological activity to BDMatrigel basement membrane matrixs, is permitted in analogue body Multigroup structure, composition, physical characteristic and function for knitting the cell basilar memebrane including liver, be conducive to hepatocyte culture and Differentiation, and the maintenance to cellular morphology and biochemical function.
As used herein, " the mature hepatocytes characteristic " includes but are not limited to one or more following indexs：1、 Express one or more hepatocyte markers, including 6 phosphatase of glucose (G6PD), albumin (Albumin), the anti-tryptoses of α- Enzyme (α -1-antitrypsin, AAT), cytokeratin 8 (CK8), cytokeratin 18 (CK18), asialoglycoprotein by Body (asialoglycoproteinreceptor, ASGR), alcohol dehydrogenase 1 (alcoholdehydrogenase1), arginine Enzyme I types (arginaseTypeI), cytochrome p450 enzyme 3A4 (CYP3A4), liver specificity organic anion transporter (LST-1) or in which combination；2, liver specificity enzymatic activity, such as G6PD and CYP3A4；The by-product of bile or urea；Removing toxic substances Function etc.；3, the characteristic form of liver cell；4, cell is proliferated in the liver of immunodeficient animals, and rebuilds liver function.
Beneficial effects of the present invention are as follows：
The invention discloses a kind of culture of personalized liver cell and amplification methods, and the method includes to hepatic parenchymal cells Genetic modification is carried out, to inhibit the expression of P53 genes, the liver cell being prepared according to the above method passes through external continuous biography 30 times or more assay certificates of generation its with unlimited multiplication capacity.
Further, the hepatic parenchymal cells after genetic modification using hepatocyte growth culture medium disclosed by the invention and Hepatocyte differentiation culture medium is cultivated and is expanded, and hepatocyte growth differentiation efficiency is improved, and extends the hepatocyte differentiation service life, The infinite multiplication for realizing liver cell overcomes the previous problem for being difficult to mass propgation in vitro and proliferation hepatic parenchymal cells, is The application of liver cell provides unlimited cell origin.
Further, the liver cell can freeze and recover repeatedly, be provided just for the storage, transhipment and use of cell Profit.
Further, there is Different Individual liver cell larger heterogeneity, method through the invention can establish by difference The liver cell library of individual hepatocyte composition, provides more comprehensively population sample for drug screening and toxicity detection, is also liver cell Personalized treatment establishes basis.
The albumin level of the hepatic parenchymal cells secretion obtained by above method culture is close to the primary adult liver parenchyma of people The level of cell Albumin Secretion, further, the cell also have selected from urea synthesis, storage glycogen, generate cell color Plain P450 enzymes carry out a phase, phase II metabolic, one or more functions in three-phase transhipment.
Description of the drawings
Fig. 1 is the form in primary cultured hepatocyt and amplification procedure, and wherein A is culture 24 hours, and B is culture 6 days, C For culture 2 weeks；
Fig. 2 is the liver cell form during differentiation and maturation, and wherein A compares for primary hepatocyte, and B trains for hepatocyte differentiation It supports 5 days, arrow show Binucleate Hepatocytes；
Fig. 3 is that RT-PCR (reverse transcription PCR) is detected through culture amplification and the liver cell of differentiation and maturation and the original of fresh separated For the expression of hepatic parenchymal cells one phase, phase II metabolic and three-phase transporter gene, RT indicates reverse transcription；
Fig. 4 is liver cell albumin (A) and CAM 120/80 (B) of the Immunofluorescence test through culture amplification and differentiation and maturation Expression；
Fig. 5 is liver cell albumin (A) of the FCM analysis through culture amplification and differentiation and maturation, and CYP3A4 (B) and α are anti- The expression of trypsase (C)；
Fig. 6 is BODIPY dyeing detection through culture amplification and the liver cell of differentiation and maturation and the primary liver parenchyma of fresh separated Fat drips in cell are horizontal；Wherein A compares for primary hepatocyte, and B is hepatocyte differentiation culture 5 days；
Fig. 7 is that the glycogen storage that PAS staining for glycogen detects in the liver cell through culture amplification and differentiation and maturation is horizontal；
Fig. 8 is that LDL-DyLight-549 takes in liver cell of the testing inspection through culture amplification and differentiation and maturation to low-density The intake ability of lipoprotein, wherein A are liver cell form under light field, and B is glimmering after corresponding to cellular uptake LDL-DyLight-549 Radiograph；
Fig. 9 is that typical medicaments lure above-mentioned cell cytochrome p 450 enzyme CYP3A4 and CYP1A2 gene expression dose It leads, wherein A is dexamethasone, and B is Omeprazole；
Figure 10 is inhibiting effect of the ketoconazole to above-mentioned cell cytochrome p 450 enzyme CYP3A4；
Figure 11 is that ELISA is detected through culture amplification and the liver cell of differentiation and maturation and the primary hepatic parenchymal cells of fresh separated The secretion level of albumin；
Figure 12 is human albumin content in the mice serum of the above-mentioned cell transplantation of detection after two weeks；
Figure 13 is the liver cell form using embodiment 2 method culture and differentiation；Wherein A is the liver cell for being proliferated form, B To break up the liver cell of form.
Specific embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.Following embodiment will be helpful to this field Technical staff further understand the present invention, but the invention is not limited in any way.It should be pointed out that the general of this field For logical technical staff, without departing from the inventive concept of the premise, various modifications and improvements can be made.These are belonged to Protection scope of the present invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition or according to Condition proposed by manufacturer.Unless otherwise stated, otherwise all percentages and parts are counted by weight.
Method therefor is conventional method unless otherwise specified in following embodiments, and agents useful for same commercially obtains .Wherein, liver cell basal medium DMEM/F12 (Invitrogen companies of the U.S., 11320-033), William'sE are (beautiful Invitrogen companies of state, A12176-01), human hepatocyte growth factor (HGF, Humanzyme companies of the U.S., HZ-1083), People's tumour inhibitor-M (OncostatinM, Peprotech companies of the U.S., 300-10), dexamethasone (Dexamethasone, the U.S. Sigma-Aldrich companies, D8893), CHIR-99021 (Selleck companies of the U.S., S1263), (U.S. Y-27632 Selleck companies, S1459), A83-01 (MCE companies, HY-10432), CollagenI (Invitrogen companies of the U.S., A1048301), Matrigel matrigels (U.S. company BD, 356234).
Embodiment 1：
One, the long-term cultivation of human primary hepatocyte and amplification
1. tissue culture plate is pre-coated.
The specific method for coating of CollagenI is as follows：Entire coating process operates on ice, will by taking 24 orifice plates coating as an example Collagen product (5mg/ml) solution is placed in spare on ice；It takes the centrifuge tube of 15ml to be placed on ice, the basis training of precooling is added Base is supported, such as：DMEM10ml；The collagen product (5mg/ml) of 100ul is taken to be added in the DMEM of above-mentioned 10ml, mixing；This Solution compound concentration is 0.05mg/ml；The above-mentioned collagen solution prepared is added in corresponding orifice plate, 24 orifice plates add per hole 300μL；Culture plate is placed on super-clean bench, it is soft to shake, so that the bottom surface that collagen solution covers entire hole is stood.Orifice plate is put in In incubator, stand overnight；Culture plate is taken out, supernatant is removed, the cleaning of 500 μ L basal mediums is added per hole twice, nature of uncapping It dries (or being dried overnight).Sealed membrane packages, 4 DEG C of preservations.In use, taking out coated good culture plate, add 500 per hole μ L cell culture complete mediums, are placed in CO2 incubators and balance 15min；Culture medium is abandoned in suction, and a certain amount of cell suspension is added, is placed in Adhere-wall culture in incubator periodically observes adherent situation.
2. the separation of the primary hepatic parenchymal cells of people.
The primary hepatic parenchymal cells of Freshman, (MaurelP., Hepatocytes- are detached using two step perfusion of clostridiopetidase A MethodsandProtocols, METHODSINMOLECULARBIOLOGY, ISSN1064-3745).
The specific method is as follows：Use PBS under the pressure that peristaltic pump provides first, the official jargon using liver surface exposure connects It is continuous to rinse fresh liver tissue 10 minutes, PBS is then changed to the Hanks perfusions hepatic tissue 10 minutes of no calcium ions and magnesium ions, Then it is filled using four Collagenase Types (Sigma Co., USA) of BSA and mass volume ratio 0.1% that mass volume ratio 1% is added Note 30 minutes.Liver cell is gently separated from hepatic tissue using glue stick, 500g is centrifuged 1 minute and is repeated 3 times, and precipitation is thin Born of the same parents are then the hepatic parenchymal cells of primary separation.
3. genetic modification
Genetic modification is carried out to the hepatic parenchymal cells of acquisition, to inhibit the expression of P53 genes；Detailed process includes：It uses Slow virus carrier loads at least one of large T antigen gene, E6E7 genes, MDM2 genes, infects the liver parenchyma of above-mentioned acquisition Cell, in the present embodiment, the ratio of infected hepatic parenchymal cells and slow virus carrier is 1：1—1：100.
4. culture and the proliferation of the primary hepatic parenchymal cells of people.
By in the incoming tissue culture plate being coated with of hepatic parenchymal cells after genetic modification, hepatocyte growth culture is added Base.Hepatocyte culture medium is that Insulin-Transferrin-sodium selenite mixing is added in basal cell culture medium DMEM/F12 to mend The small molecule of filling liquid, GlutaMAX, nonessential amino acid, beta -mercaptoethanol, human hepatocyte growth factor, Wnt signal paths swashs Dynamic agent CHIR99021, Rho related protein kinase enzyme inhibitor Y-27632, ALK5 kinase inhibitor A83-01.Hepatocyte growth is trained It supports in base, Insulin-Transferrin-sodium selenite mix supplement liquid final concentration of 1% (volumn concentration)；Human liver cell is given birth to The final concentration 20ng/ml of the long factor；Final concentration of 3 μM of the small molecule agonist CHIR99021 of Wnt signal paths；Rho is related Final concentration of 10 μM of kinases inhibitor Y-27632；Final concentration of 1 μM of ALK5 kinase inhibitors A83-01；β-sulfydryl The final concentration of 0.1mM of ethyl alcohol；Final concentration of 1% (volumn concentration) of nonessential amino acid；The final concentration of GlutaMAX For 1% (volumn concentration).Replace fresh hepatocyte growth culture medium within every 3 days in cell cultivation process.Cell passage Method is to digest cell with Accutase digestive juices (Invitrogen companies of the U.S.), by 1:2-1:5 passages, are increased with liver cell Grow medium culture.
Two, the identification of the differentiation and maturation and cellularity of liver cell
1. differentiation and the maturation of liver cell.
Hepatic cell growth to 90% converge rate when, hepatocyte differentiation medium culture is added 9-13 days, replaces daily fresh Hepatocyte differentiation culture medium.Hepatocyte differentiation culture medium is that insulin-is added in basal cell culture medium DMEM/F12 to turn iron Albumen-sodium selenite mix supplement liquid, GlutaMAX, nonessential amino acid, beta -mercaptoethanol, dexamethasone and people's tumour inhibitor- M；In hepatocyte differentiation culture medium, the final concentration of 1% (volume basis of Insulin-Transferrin-sodium selenite mix supplement liquid Content), final concentration of 1% (volumn concentration) of GlutaMAX, final concentration of 0.1 μM of dexamethasone, people's tumour inhibitor-M Final concentration of 10ng/ml, final concentration of 10 μM of ALK5 kinase inhibitors SB431542, cell is cultivated in differential medium There is within 3-5 days hepatic parenchymal cells form (see Fig. 2).
2. hepatocyte markers and Function Identification.
The liver cell of the liver cell of present invention culture amplification and differentiation and maturation is subjected to expression and function of genes identification respectively, Specific method and result are as follows：
1)RT-PCR：Prompt biology RNAfast200 kits (article No. 220010) are flown using Shanghai and extract above-mentioned cell RNA, Using SuperScriptTMIIRNaseH- reverse transcriptase (Invitorgen, 18064014) by RNA reverse transcriptions be cDNA, use Primer in table one expands liver cell related gene by round pcr, carries out gel electrophoresis, and qualification result is shown in Fig. 3, specially by this The liver cell of sharp method culture and proliferation can express the gene closely related with hepatic parenchymal cells function after vitro differentiation, Expression is close with primary hepatic parenchymal cells.
2) immunofluorescence：Above-mentioned cell fixes 10 minutes through 4% paraformaldehyde room temperature, and film 10 is worn with 0.2%Triton100 Minute, it is closed 1 hour with 1%BSA room temperatures, 4 DEG C of overnight incubations of primary antibody is added, PBS is washed three times, and it is small that secondary antibody incubation at room temperature 1 is added When, inverted microscope observation imaging, qualification result are shown in Fig. 4 after PBS washings three times, reflect hepatic parenchymal cells function albumin and The CAM 120/80 of epithelial cell characteristic can be detected.
3) flow cytometry：Using eBioscience fluidic cell staining kits, detected according to kit specification Albumin in cell, the expression of CYP3A4 and alpha-1 antitrypsin are stated, testing result is shown in Fig. 5, above-mentioned three kinds of reflections liver parenchyma The albumen of cell function it is most of through the liver cell of differentiation in express, further prove the liver cell tool that this patent method obtains There are higher purity and function.
4) BODIPY is dyed：Horizontal, the inspection using the above-mentioned intracellular fat drips of BODIPY detections of Invitrogen companies production It surveys result and sees that Fig. 6 is compared with primary hepatic parenchymal cells, the liver cell through in vitro culture and differentiation also has the energy of synthesis fat drips Power.
5) glycogen PAS is dyed：The staining for glycogen kit that company's production is built up using Nanjing, is examined according to kit specification It surveys above-mentioned cell Glycogen synthesis and storage is horizontal, testing result is shown in Fig. 7, and the liver cell through in vitro culture and differentiation has stronger Glycogen synthesis ability reflects its peculiar function with hepatic parenchymal cells.
6) LDL intakes experiment：Kit is absorbed using the low-density lipoprotein (LDL) of Abcam companies production (ab133127), intake ability of the above-mentioned cell to LDL is detected according to kit specification, testing result is shown in Fig. 8, through training in vitro It supports and the liver cell of differentiation has the ability for absorbing LDL, reflect its peculiar function with hepatic parenchymal cells.
7) cytochrome oxidase induces：It is handled respectively using 100 μM of dexamethasone and 10 μM above-mentioned through cultivating amplification simultaneously The liver cell of differentiation and maturation, using above-mentioned above-mentioned using winged prompt biology RNAfast200 kits (article No. 220010) extracting in Shanghai RNA reverse transcriptions are by cell RNA using SuperScriptTMIIRNaseH- reverse transcriptase (Invitorgen, 18064014) CDNA detects the table of cytochrome oxidase CYP3A4 and CYP1A2 using the primer in table one by Real-time quantitative PCR Change up to level.Testing result is shown in Fig. 9, it is known that clinically used some drugs such as dexamethasone and Omeprazole are passed through in liver Specific cytochrome oxidase (CYP) is converted and is metabolized, and Fig. 9 A show that dexamethasone can significantly inducing cell CYP3A4 Transcriptional level increases, and CYP1A2 then can be by the notable induced expression of Omeprazole.
8) cytochrome oxidase Inhibition test uses the CYP3A4P450-GLOTM (V9001) of Promega companies production Kit detects the specific inhibitor ketoconazole of CYP3A4 to the active inhibiting effect of CYP3A4, inspection according to kit specification The result is shown in Figure 10 is surveyed, CYP3A4 activity can be significantly inhibited by ketoconazole, and result above confirms, the liver of this patent culture amplification is thin Born of the same parents have typical hepatic parenchymal cells CYP activity after vitro differentiation.
9)ELISA：It is detected in above-mentioned cells and supernatant using the human albumin ELISA kit of Bethyl companies production Albumin content, testing result is shown in that Figure 11 is compared with primary hepatic parenchymal cells, and the liver cell through in vitro culture and differentiation has Stronger albumin synthesis capability reflects its peculiar function with hepatic parenchymal cells.
10) cell transplantation and the detection of serum human albumin：By it is above-mentioned through culture amplification and differentiation and maturation liver cell 2 × 106 cells are drawn blood, are given birth to using Bethyl companies by being injected into Fah-/-/il2R-/-/Rag2-/- mouse spleen after two weeks The human albumin ELISA kit of production detects human albumin level in mice serum, detection according to the specification that kit provides The result is shown in Figure 12 can detect human albumin in two weeks or so in transplanting in Mice Body, prompt the liver cell of transplanting successfully fixed It is implanted into the liver of Recipient mice.
Embodiment 2：
1. tissue culture plate is pre-coated.
It is coated with using Matrigel, the Matrigel of freezen protective is placed 4 DEG C overnight, liquid is become, uses The serum free medium (such as DMEM) of precooling presses 1:80 dilutions, are added in culture hole, are advisable with covering bottom surface, place 37 DEG C one small When after can be used.
2. the separation of the primary hepatic parenchymal cells of people is the same as embodiment 1.
3. genetic modification
At least one of large T antigen gene, E6E7 genes or MDM2 genes are loaded using carrier for expression of eukaryon, using normal Rule transfection reagent transfects the hepatic parenchymal cells of above-mentioned acquisition.
4. culture and the proliferation of the hepatic parenchymal cells after genetic modification.
The final concentration 20ng/ml of human hepatocyte growth factor in above-mentioned hepatocyte growth culture medium；Wnt signal paths Final concentration of 3 μM of small molecule agonist CHIR99021；Final concentration of 10 μ of Rho related protein kinase enzyme inhibitors Y-27632 Final concentration of 1 μM of M, ALK5 kinase inhibitor A83-01, remaining is the same as embodiment 1.
With embodiment 1, qualification result is consistent with embodiment 1 for the identification of the differentiation and maturation and cellularity of liver cell.
Effect identical with 1 method of embodiment, cell Proliferation and differentiation form and reality are obtained using the method for embodiment 2 It is identical to apply the cell that 1 method of example is obtained.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this The principle of invention, various changes and improvements may be made to the invention without departing from the spirit and scope of the present invention, these changes Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its Equivalent defines.
Those skilled in the art are supplied to the purpose described to the description of the various embodiments of the present invention above.It is not It is intended to exhaustive or is not intended to and limits the invention to single disclosed embodiment.As described above, the present invention's is various It substitutes and variation will be apparent for above-mentioned technology one of ordinary skill in the art.Therefore, although specifically begging for Some alternative embodiments are discussed, but other embodiment will be apparent or those skilled in the art are opposite It is easy to obtain.The present invention is intended to include all replacements of the present invention crossed by discussion herein, modification and variations, and fall Other embodiment in the spirit and scope of above-mentioned application.
Although depicting the present invention by embodiment, it will be appreciated by the skilled addressee that there are many deformations by the present invention With variation without departing from the spirit of the present invention, it is desirable to which the attached claims include these deformations and change without departing from the present invention Spirit.

Claims (18)

1. culture and the amplification method of personalized liver cell, which is characterized in that include the following steps：
Step 1：Genetic modification is carried out to the hepatic parenchymal cells of acquisition, to inhibit expression or the function of P53 genes；
Step 2：Will in step 1 by genetic modification hepatic parenchymal cells merging hepatocyte growth culture medium in carry out culture and Amplification obtains liver cell；
Step 3：The liver cell that step 2 is obtained is placed in culture in hepatocyte differentiation culture medium and obtains mature hepatocytes.
2. culture and the amplification method of personalization liver cell according to claim 1, which is characterized in that institute in step 1 The method that the hepatic parenchymal cells stated carry out genetic modification is selected from least one of channel genes, gene editing or RNA interference.
3. culture and the amplification method of personalization liver cell according to claim 2, which is characterized in that the channel genes The gene used is selected from the gene for inhibiting P53 expression or function, described that the gene of P53 expression or function is inhibited to be selected from SV40 viruses Large T antigen gene, HPV viruse E6E7 genes or ubiquitin ligase gene；The ubiquitin ligase gene be selected from Mdm2, Hdm2, At least one of COP1, Pirh2 or ARF-BP1 gene, the gene editing are selected from CRISPR/Cas9, TALEN or ZFN At least one of method.
4. culture and the amplification method of personalization liver cell according to claim 2, which is characterized in that the channel genes Method be selected from viral vectors mediate gene expression or non-virus carrier mediate gene expression.
5. culture and the amplification method of personalization liver cell according to claim 1, which is characterized in that the liver parenchyma Cell is mammal hepatic parenchymal cells, and the mammal hepatic parenchymal cells are selected from mouse hepatocytes, rat liver parenchyma It is one or more in cell, rhesus macaque hepatic parenchymal cells, orangutan hepatic parenchymal cells or people's hepatic parenchymal cells.
6. culture and the amplification method of personalization liver cell according to claim 1, which is characterized in that described to pass through gene The hepatic parenchymal cells of modification are attached on culture support, add what hepatocyte growth culture medium was cultivated and expanded, The culture support is pre-coated using collagen or extracellular matrix protein；The extracellular matrix protein is selected from Matrigel matrigels, the culture support refer to culture dish or culture plate.
7. culture and the amplification method of personalization liver cell according to claim 1, which is characterized in that the liver cell Proliferated culture medium includes basal medium, the basal medium be selected from MEM, DMEM, BME, DMEM/F12, RPMI1640, At least one of WilliamE, NeurobasalE or Fischers culture medium.
8. culture and the amplification method of personalization liver cell according to claim 7, which is characterized in that the liver cell Culture medium further includes nutritional ingredient, the nutritional ingredient include serum, serum substitute, growth factor, Wnt signal paths swash At least one of dynamic agent, Rho related protein kinases enzyme inhibitor, ALK5 kinase inhibitors.
9. culture and the amplification method of personalization liver cell according to claim 8, which is characterized in that the serum replaces It is selected from KnockOut for objectTMSerum substitute, Insulin-Transferrin-sodium selenite mix supplement liquid, N2 nutritional supplements, At least one of B27 nutritional supplements；The growth factor is selected from epidermal growth factor, fibroblast growth factor- 2, at least one of hepatocyte growth factor, platelet cell growth factor or insulin-like growth factor；The Wnt accesses Agonist is selected from least one of CHIRP98014, TWS119, CHIRP99021, Wnt3a or R-Rspondin；Described Rho related protein kinase enzyme inhibitors are selected from least one of thiazovivin, Y-27632 or Blebbistatin；Described ALK5 kinase inhibitors are selected from least one of SB431542, A83-01 or RepSox.
10. culture and the amplification method of personalization liver cell according to claim 1, which is characterized in that the liver is thin Born of the same parents' differential medium is made of basal cell culture medium and additive, the basal cell culture medium be selected from MEM, DMEM, BME, At least one of DMEM/F12, RPMI1640, William E, Neurobasa l or Fischers culture mediums；Described adds Add object include in serum substitute, hepatocyte growth factor, dexamethasone, people's tumour inhibitor-M or ALK5 kinase inhibitor at least It is a kind of.The ALK5 kinase inhibitors are selected from least one of SB431542, A83-01 or RepSox.
11. culture and the amplification method of personalization liver cell according to claim 1, which is characterized in that the step 2 Further include：Secondary culture and amplification are carried out to the liver cell after cultivating and expand 7-10 days.
12. the liver being prepared according to the culture of claim 1 to 11 any one of them personalization liver cell and amplification method Cell.
13. application of the liver cell according to claim 12 in establishing personalized liver cell library.
14. application of the liver cell according to claim 12 in compound and the toxicology and pharmacological evaluation of drug.
15. application of the liver cell according to claim 12 in preparing diagnosis or treatment hepatitis virus medicament.
16. application of the liver cell according to claim 12 in preparing bioartificial liver's material.
17. application of the liver cell according to claim 12 in hepatocyte transplantation.
18. application of the liver cell according to claim 12 in the preparation of humanization Chi-meric mice.
CN201710172028.7A 2017-03-22 2017-03-22 The culture of personalized liver cell and amplification method and its application Pending CN108330099A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710172028.7A CN108330099A (en) 2017-03-22 2017-03-22 The culture of personalized liver cell and amplification method and its application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710172028.7A CN108330099A (en) 2017-03-22 2017-03-22 The culture of personalized liver cell and amplification method and its application

Publications (1)

Publication Number Publication Date
CN108330099A true CN108330099A (en) 2018-07-27

Family

ID=62922219

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710172028.7A Pending CN108330099A (en) 2017-03-22 2017-03-22 The culture of personalized liver cell and amplification method and its application

Country Status (1)

Country Link
CN (1) CN108330099A (en)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109337858A (en) * 2018-09-20 2019-02-15 中国人民解放军第二军医大学 The liver precursor like cell model in the primary hepatocyte source for hepatitis B virus infection, preparation method and application
WO2020056661A1 (en) * 2018-09-20 2020-03-26 赵永祥 Method for fast gene editing and constructing primate disease model
CN111206013A (en) * 2020-02-13 2020-05-29 上海交通大学医学院附属仁济医院 Culture method for maintaining functional state of human primary hepatocytes for long time
CN111808795A (en) * 2020-06-28 2020-10-23 广东省医疗器械研究所 Hepatocyte in-vitro co-culture system and construction method and application thereof
CN112094803A (en) * 2020-03-18 2020-12-18 上海科技大学 Hepatocyte culture medium, culture method and application thereof
WO2021004129A1 (en) 2019-07-11 2021-01-14 上海赛立维生物科技有限公司 Method for constructing hepatic progenitor cell-like cell bank, cell lines prepared therefrom and application thereof
WO2021204061A1 (en) 2020-04-09 2021-10-14 上海赛立维生物科技有限公司 Amplification and differentiation method for pancreatic cells and use thereof
CN113564098A (en) * 2021-06-15 2021-10-29 广东乾晖生物科技有限公司 Culture method for enhancing hepatocyte functionality and hepatocyte culture solution used in culture method
CN117431203A (en) * 2023-12-20 2024-01-23 苏州瑞徕生物科技有限公司 Culture additive for enhancing expression of in vitro cultured hepatocyte coagulation factor and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104830906A (en) * 2014-02-12 2015-08-12 北京维通达生物技术有限公司 Method of obtaining functionalized human liver parenchymal cell through re-programming
CN105296418A (en) * 2014-08-04 2016-02-03 上海赛立维生物科技有限公司 Method for long-time in-vitro culturing and proliferating hepatic cells and application of method

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104830906A (en) * 2014-02-12 2015-08-12 北京维通达生物技术有限公司 Method of obtaining functionalized human liver parenchymal cell through re-programming
CN105296418A (en) * 2014-08-04 2016-02-03 上海赛立维生物科技有限公司 Method for long-time in-vitro culturing and proliferating hepatic cells and application of method

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
JANE A. BOND ET AL.: "Mutant p53 rescues human diploid cells from senescence without inhibiting the induction of SDI1/WAF1", 《CANCER RESEARCH》 *
牛育鸿等: "肿瘤抑制基因与衰老的研究进展", 《基因组学与应用生物学》 *
赵思达等: "p53与细胞衰老关系的研究进展", 《诊断学理论与实践》 *
魏如雪等: "抑制体细胞衰老促进诱导性多潜能干细胞（iPSC）生成的研究进展", 《中国农业科学》 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109337858B (en) * 2018-09-20 2022-03-15 中国人民解放军第二军医大学 Liver precursor sample cell model of primary hepatocyte source for hepatitis B virus infection, preparation method and application
WO2020056661A1 (en) * 2018-09-20 2020-03-26 赵永祥 Method for fast gene editing and constructing primate disease model
CN109337858A (en) * 2018-09-20 2019-02-15 中国人民解放军第二军医大学 The liver precursor like cell model in the primary hepatocyte source for hepatitis B virus infection, preparation method and application
WO2021004129A1 (en) 2019-07-11 2021-01-14 上海赛立维生物科技有限公司 Method for constructing hepatic progenitor cell-like cell bank, cell lines prepared therefrom and application thereof
CN111206013A (en) * 2020-02-13 2020-05-29 上海交通大学医学院附属仁济医院 Culture method for maintaining functional state of human primary hepatocytes for long time
CN112094803A (en) * 2020-03-18 2020-12-18 上海科技大学 Hepatocyte culture medium, culture method and application thereof
CN112094803B (en) * 2020-03-18 2022-08-02 上海科技大学 Hepatocyte culture medium, culture method and application thereof
WO2021204061A1 (en) 2020-04-09 2021-10-14 上海赛立维生物科技有限公司 Amplification and differentiation method for pancreatic cells and use thereof
CN111808795A (en) * 2020-06-28 2020-10-23 广东省医疗器械研究所 Hepatocyte in-vitro co-culture system and construction method and application thereof
CN113564098B (en) * 2021-06-15 2022-07-08 广东乾晖生物科技有限公司 Culture method for enhancing hepatocyte functionality and hepatocyte culture solution used in culture method
CN113564098A (en) * 2021-06-15 2021-10-29 广东乾晖生物科技有限公司 Culture method for enhancing hepatocyte functionality and hepatocyte culture solution used in culture method
CN117431203A (en) * 2023-12-20 2024-01-23 苏州瑞徕生物科技有限公司 Culture additive for enhancing expression of in vitro cultured hepatocyte coagulation factor and application thereof
CN117431203B (en) * 2023-12-20 2024-03-22 苏州瑞徕生物科技有限公司 Culture additive for enhancing expression of in vitro cultured hepatocyte coagulation factor and application thereof

Similar Documents

Publication Publication Date Title
CN108330099A (en) The culture of personalized liver cell and amplification method and its application
CN105296418B (en) A kind of method and its application of long-term in vitro culture and amplification liver cell
US9931360B2 (en) Isolated liver stem cells
CN110317775B (en) Culture medium for hepatocyte culture and liver organoid preparation
US20200354683A1 (en) Derivation of hepatic stem cells and mature liver cell types and uses thereof
Shulman et al. Long-term culture and coculture of primary rat and human hepatocytes
US10457914B2 (en) Optimized methods for differentiation of cells into cells with hepatocyte and hepatocyte progenitor phenotypes, cells produced by the methods, and methods for using the cells
EP3397753B1 (en) Microtissue formation using stem cell-derived human hepatocytes
Kim et al. Enhanced metabolizing activity of human ES cell-derived hepatocytes using a 3D culture system with repeated exposures to xenobiotics
JP2017099417A (en) Bioartificial proximal tubule systems and methods of using the same
Gu et al. Establishment of a three‐dimensional co‐culture system by porcine hepatocytes and bone marrow mesenchymal stem cells in vitro
WO2011016485A1 (en) METHOD FOR INDUCING DIFFERENTIATION OF iPS CELLS INTO HEPATIC PARENCHYMAL CELLS
Cohen et al. Coculture and long-term maintenance of hepatocytes
Furth et al. Stem cell populations giving rise to liver, biliary tree, and pancreas
US20220160785A1 (en) Method for manufacturing cell population including liver precursor cells
RU2663118C1 (en) Cell product of insulin-producing mammalian cells and use thereof for diabetes mellitus therapy
Pettinato et al. Human embryoid bodies to hepatocyte-like clusters: Preparing for translation
Deng Microfabricated 3D Culture for Promoting Hepatic Differentiation of Stem Cell and Maintaining Hepatocyte Function for Liver Tissue Engineering
BRPI0620049A2 (en) isolated liver stem cells
Ogoke Bioengineered 3D Models of Hepatic Cell Migration for Applications in Liver Regenerative Medicine
Rebelo Development of 3D in vitro models for prediction of hepatic metabolism and toxicity
Ulrich et al. Pancreatic differentiation from human pluripotent stem cells
Bierwolf et al. Liver tissue engineering
Omer et al. Human embryo pancreatic stem cells differentiating into active insulin secreting islet-like structure

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
EE01 Entry into force of recordation of patent licensing contract
EE01 Entry into force of recordation of patent licensing contract

Application publication date: 20180727

Assignee: Shanghai Selvie Medical Technology Co., Ltd.

Assignor: SHANGHAI SAILIWEI BIOTECHNOLOGY CO., LTD.

Contract record no.: X2019310000001

Denomination of invention: Culture and amplification method and applications of personalized hepatocytes

License type: Common License

Record date: 20190813

WD01 Invention patent application deemed withdrawn after publication
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20180727