WO2021004129A1 - 肝祖细胞样细胞库的构建方法及其制备的细胞株与应用 - Google Patents

肝祖细胞样细胞库的构建方法及其制备的细胞株与应用 Download PDF

Info

Publication number
WO2021004129A1
WO2021004129A1 PCT/CN2020/086659 CN2020086659W WO2021004129A1 WO 2021004129 A1 WO2021004129 A1 WO 2021004129A1 CN 2020086659 W CN2020086659 W CN 2020086659W WO 2021004129 A1 WO2021004129 A1 WO 2021004129A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
culture
hepatic progenitor
cell line
donor
Prior art date
Application number
PCT/CN2020/086659
Other languages
English (en)
French (fr)
Inventor
鄢和新
Original Assignee
上海赛立维生物科技有限公司
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 上海赛立维生物科技有限公司 filed Critical 上海赛立维生物科技有限公司
Priority to EP20830058.2A priority Critical patent/EP3978600A4/en
Priority to US17/622,838 priority patent/US20220411758A1/en
Publication of WO2021004129A1 publication Critical patent/WO2021004129A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • C12N5/0672—Stem cells; Progenitor cells; Precursor cells; Oval 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • 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
    • 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
    • C12N5/0671—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
    • 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
    • G01N33/5014—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 for testing toxicity
    • 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
    • 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
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • 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
    • C12N2510/04—Immortalised 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to the field of biotechnology, in particular to a method for constructing a liver progenitor cell-like cell bank, and cell lines prepared and applications thereof.
  • Liver failure is the end-stage manifestation of severe liver disease, and the mortality rate of patients can be as high as 50% to 90%. Among them, the problem of drug-induced liver injury caused by adverse drug reactions in patients is more prominent, accounting for more than 50% of acute liver failure cases, resulting in drug development failure or withdrawal after marketing.
  • hepatocytes Most of the reasons for drug development failure or withdrawal after marketing are due to specific toxicity to patients.
  • the immortalization of hepatocytes is one of the important ways to solve the source of hepatocytes.
  • primary hepatocytes or hepatocyte-like cells derived from induced stem cells are immortalized, and the obtained cell lines are applied to the liver. Metabolic heterogeneity studies to find solutions to drug-specific toxicity problems.
  • the Chinese invention patent application with publication number CN108330099A discloses a method for culturing and amplifying individualized hepatocytes.
  • the method puts genetically modified hepatocytes in a hepatocyte proliferation medium for culturing and expansion, and then The obtained hepatocytes were differentiated and cultured to obtain mature hepatocytes.
  • this method firstly genetically modifies the hepatocytes, and then carries out the subsequent proliferation and differentiation culture, which easily affects the in vitro proliferation ability of the mature hepatocytes obtained, and the source of the donor is single, which makes it impossible to carry out drug-specific toxicity studies.
  • the purpose of the present invention is to provide a method for constructing a liver progenitor cell-like cell bank and the prepared cell lines and applications, so as to obtain immortal hepatic progenitor cell-like cell lines with good proliferation ability in vitro, and to facilitate the development of drug-specific toxicity the study.
  • the method for constructing the liver progenitor-like cell bank of the present invention includes:
  • S1 Provide human primary hepatocyte cultures from different donors, and conduct human primary hepatocyte cultures from each donor at a seeding density of 0.5 ⁇ 10 4 -5 ⁇ 10 4 cells/cm27 -14 days of transformation culture, and then freezing the liver progenitor cell-like cell lines from each donor obtained after the transformation culture to obtain a heterogeneous hepatic progenitor cell-like cell bank;
  • S2 Thaw the hepatic progenitor-like cell line from each donor and carry out proliferation and culture to obtain adherent cells from different donors.
  • the confluence rate of adherent cells from each donor is 70%-90 %;
  • S3 Perform the first passaging treatment and virus infection on the adherent cells from each donor in sequence, and perform medium replacement during the virus infection;
  • S4 Perform a second passage with a number of passages of 2 or 3 on each donor-derived culture obtained after infection with the virus, and perform a second passage of the culture obtained after the second passage by selecting a medium Continuous screening and culture to obtain infection cultures from different donor sources;
  • S5 Perform continuous subculture with a passage ratio of 1:2-1:4 and a number of passages of 5-10 to each donor-derived infection culture to obtain the hepatic progenitor-like cell bank.
  • the beneficial effect of the method for constructing the hepatic progenitor-like cell bank of the present invention is that in the step S11, the number of human primary hepatocyte cultures from each donor is 0.5 ⁇ 10 4 -5 ⁇ 10 4 Transformation culture for 7-14 days at a seeding density of 7-14 days is conducive to imparting good proliferation performance to the human primary hepatocyte culture, combined with the subsequent treatment of the second passage, the screening treatment and the The control of continuous subculture has resulted in immortal hepatic progenitor cell-like cell lines from different donor sources with good proliferation ability in vitro, which is conducive to the development of drug-specific toxicity studies.
  • the culture obtained after the transformation culture is sequentially subjected to 2 or 3 expansion cultures, and then the freezing treatment is performed.
  • the beneficial effect is that it further endows the human primary hepatocyte culture with good proliferation performance.
  • the hepatic progenitor cell-like cell line undergoes the proliferation culture at a seeding density of 0.5 ⁇ 10 4 -5 ⁇ 10 4 cells/cm2, and within three days after the start of the proliferation culture , The medium is replaced every day.
  • the beneficial effect is that the adherent cells can be obtained as soon as possible while ensuring cell viability.
  • the adherent cells derived from each donor are subjected to the first passage treatment for 24 hours at a seeding density of 2 ⁇ 10 4 -4 ⁇ 10 4 cells/cm 2.
  • the beneficial effect is that the proper inoculation density is beneficial to the effective subsequent virus infection.
  • the culture obtained after the first passage treatment is infected with the virus at an inoculation density of 2 ⁇ 10 4 to 4 ⁇ 10 4 cells/cm 2.
  • the beneficial effect is that a proper inoculation density is beneficial to the effective progress of virus infection.
  • the ratio of the number of the lentivirus to the adherent cells is 0.5-60.
  • each donor-derived culture obtained after infection with the virus is subjected to an inoculation density of 2 ⁇ 10 4 -4 ⁇ 10 4 cells/cm 2 for 5-7 days.
  • the culture medium is replaced every 2-3 days during the second passage treatment.
  • the screening medium is a TEM medium.
  • the infection culture from each donor is subjected to the continuous subculture at an inoculation density of 2 ⁇ 10 4 -4 ⁇ 10 4 cells/cm 2.
  • the immortal hepatic progenitor cell-like cell lines in the heterogeneous immortal hepatic progenitor cell-like cell bank prepared by the construction method have good in vitro proliferation ability, in vitro construction of the heterogeneous immortal hepatic progenitor cell-like cell bank
  • the three-dimensional hepatocyte model is then used to detect the specific hepatotoxicity of the drug by using the three-dimensional hepatocyte model in vitro, which is conducive to the research on the specific toxicity of the drug.
  • the immortal liver progenitor cell-like cell line prepared by the construction method can be applied to bioartificial liver and liver cell transplantation.
  • the present invention also provides an immortal hepatic progenitor cell-like cell line classified as 81.5 prepared by using the construction method, and the immortal hepatic progenitor cell-like cell line is deposited in the China Type Culture Collection in Wuhan University, Wuhan, China , The deposit number is CCTCC NO: C2019125.
  • the Chinese Type Culture Collection received the culture on June 11, 2019 and named it the immortalized human hepatic progenitor cell line ALI-CELL-81.5, and tested and identified the culture on June 24, 2019 The thing is alive.
  • the immortalized human liver progenitor cell-like cell line ALI-CELL-81.5 is the immortal liver progenitor cell-like cell line classified as 81.5 according to the present invention.
  • the China Type Culture Collection shall, upon request, store the culture for 30 years starting from June U, 2019, and shall continue to store it for another 5 years after receiving the request to provide culture samples before the expiration date.
  • the present invention also provides the application of the immortal liver progenitor cell-like cell line in bioartificial liver and liver cell transplantation, and the hepatocytes formed by the proliferation of the immortal hepatic progenitor cell line are secreted during the treatment of the bioartificial liver. Exogenous human growth factor.
  • the at least one exogenous human growth factor includes any one or more of human hepatocyte growth factor, human transforming growth factor- ⁇ , and human interleukin-6.
  • Figure 1 is a schematic diagram of the morphology of the human primary hepatocytes of the present invention.
  • Figure 2 is a schematic diagram of the morphology of the first hepatic progenitor-like cell line of the present invention
  • Fig. 3 is a comparison diagram of the proliferation performance of the first immortal hepatic progenitor cell-like cell line, the second immortal hepatic progenitor cell-like cell line, the third immortal hepatic progenitor cell line and the fourth immortal hepatic progenitor cell line of the present invention ;
  • Figure 4a is a schematic diagram of the morphology of the first immortal hepatic progenitor cell line of the present invention after proliferation and culture in vitro;
  • Figure 4b is a schematic diagram of the morphology of the second immortal hepatic progenitor cell line of the present invention after proliferation and culture in vitro;
  • Figure 4c is a schematic diagram of the morphology of the third immortal hepatic progenitor cell line of the present invention after proliferation and culture in vitro;
  • Figure 4d is a schematic diagram of the morphology of the fourth immortal hepatic progenitor cell line of the present invention after proliferation and culture in vitro;
  • Figure 5 is a schematic diagram of the morphology of the first hepatocyte model strain of the present invention.
  • Figure 6 a Schematic diagram of the specific hepatotoxicity of Erlotinib in the first hepatocyte model strain of the present invention
  • Figure 6b is a schematic diagram of the specific hepatotoxicity of Erlotinib of the second hepatocyte model strain of the present invention.
  • Fig. 6c is a schematic diagram of the specific hepatotoxicity of Erlotinib of the third hepatocyte model strain of the present invention.
  • Fig. 6d is a schematic diagram of the specific hepatotoxicity of Erlotinib of the fourth hepatocyte model strain of the present invention.
  • Figure 6e is a schematic diagram of the specific hepatotoxicity of Erlotinib of the fifth hepatocyte model strain of the present invention.
  • Figure 6f is a schematic diagram of the specific hepatotoxicity of Erlotinib in the sixth hepatocyte model strain of the present invention.
  • Figure 7 is a schematic structural diagram of the liquid-gas interactive bioreactor of the present invention.
  • Fig. 8 is a schematic diagram of the morphology of the proliferating cells obtained after cell proliferation of the present invention observed under a cell inverted microscope at a magnification of 100 times;
  • Figure 9 is a schematic diagram of the structure of the extracorporeal circulation system of the present invention.
  • Figure 10 is an electron micrograph of the cell ball microcapsule of the present invention.
  • the TEM culture medium is from Shanghai Sailiwei Biotechnology Co., Ltd.; Matrigel is produced by Corning, the catalog number is 356234; the polybrene suspension is produced by Shanghai Yisheng Biological Technology Co., Ltd., the catalog number is 40804ES76; Pancreatin Digestion solution, DMEM/12 medium and HepX medium are produced by Shanghai Yuanpei Biotechnology Co., Ltd., and the product numbers are S310KJ, L110KJ and X071A1, respectively.
  • the cell culture plates with different numbers of holes and the culture dishes with different diameters were all produced by NEST Science Co. Ltd.
  • the cell culture incubator was purchased from ESCO Singapore, the product model is CLL- 170B-8; the cell inverted microscope was purchased from Nikon Co., Ltd., and the product model was Ta2-FL.
  • embodiments of the present invention provide a method for constructing a hepatic progenitor-like cell bank, including:
  • S1 Provide human primary hepatocyte cultures from different donors, and conduct human primary hepatocyte cultures from each donor at a seeding density of 0.5 ⁇ 10 4 -5 ⁇ 10 4 cells/cm27 -14 days of transformation culture, and then freezing the liver progenitor cell-like cell lines from each donor obtained after the transformation culture to obtain a heterogeneous hepatic progenitor cell-like cell bank;
  • S2 Thaw the hepatic progenitor-like cell line from each donor and carry out proliferation and culture to obtain adherent cells from different donors.
  • the confluence rate of adherent cells from each donor is 70%-90 %;
  • S3 Perform the first passaging treatment and virus infection on the adherent cells from each donor in sequence, and perform medium replacement during the virus infection;
  • S4 Perform a second passage with a number of passages of 2 or 3 on each donor-derived culture obtained after infection with the virus, and perform a second passage of the culture obtained after the second passage by selecting a medium Continuous screening and culture to obtain infection cultures from different donor sources;
  • S4 Perform continuous subculture with a passage ratio of 1:2-1:4 and a number of passages of 5-10 to each donor-derived infection culture to obtain the hepatic progenitor-like cell bank.
  • the hepatic progenitor-like cell bank in the embodiment of the present invention is a heterogeneous immortalized hepatic progenitor-like cell bank.
  • the cryopreservation treatment is performed using cell cryopreservation liquid and liquid nitrogen.
  • the virus used for the virus infection is recorded in the Chinese invention patent application with publication number CN108330099A. Specifically, it expresses the SV40 virus large T antigen gene, HPV virus E6E7 gene or ubiquitin ligase gene.
  • the selection medium is TEM medium.
  • the culture obtained after the transformation culture is sequentially subjected to 2-3 times of expansion culture and the freezing treatment.
  • the hepatic progenitor-like cell line is subjected to the proliferation culture at a seeding density of 0.5 ⁇ 10 4 -5 ⁇ 10 4 cells/cm 2, and after the start of the proliferation culture During the three days, the culture medium was replaced every day.
  • the immortal hepatic progenitor cell-like cell strain prepared by the construction method can be applied to bioartificial liver, hepatocyte transplantation, and specific hepatotoxicity detection of drugs.
  • This example provides the first hepatic progenitor-like cell line, classified and named as 81.5, deposited in the Chinese Type Culture Collection Center, and the first immortal hepatic progenitor-like cell line with the deposit number of CCTCC NO: C2019125, and the A method for constructing immortal liver progenitor cell line.
  • the coating solution in this embodiment is a mixed solution of HepX medium and Matrigel, and the volume ratio of the HepX medium to the Matrigel is 80:1.
  • the incubator is a 6-well cell culture plate
  • 250-350 microliters of coating solution is added to each culture well to perform the coating treatment in advance.
  • the incubator is a 12-well cell culture plate
  • the incubator is a cell culture dish with a diameter of 6 cm, 500-700 microliters of coating solution is added for coating treatment.
  • the incubator is a cell culture dish with a diameter of 10 cm, add 1000-1500 microliters of coating solution for coating.
  • the incubator is a cell culture dish with a diameter of 15 cm, and 2000-3000 microliters of coating solution is added for coating treatment.
  • the coating processing time is 30-120 minutes.
  • the cell culture incubator involved in this embodiment has a constant temperature of 37° C., saturated humidity, and a carbon dioxide concentration of 5%. Except for the process carried out in the cell culture incubator, other operations are carried out in a normal temperature aseptic operation table.
  • the cell digestion solution is trypsin digestion solution.
  • the method for constructing the first immortal hepatic progenitor-like cell line is specifically:
  • a first human primary hepatocyte culture is provided, and the first human primary hepatocyte culture is transformed and cultured at a seeding density of 1 ⁇ 10 4 cells/cm 2 for 7 days, and then The first hepatic progenitor cell-like cell line obtained after the transformation and culture is sequentially subjected to 3 expansion treatments.
  • any one of the two-step perfusion method or the chopping digestion method is used to separate the tissue adjacent to the human hepatic hemangioma derived from the first donor to obtain the first human primary hepatocyte culture
  • the specific implementation of the two-step perfusion method was recorded in the "Expansion and differentiation of human hepatocyte-derived liver progenitor-like cells for the study of hepatotropic pathogens" published in the first issue of Volume 29 of Cell Research in 2018.
  • the specific implementation of the shredding and digestion method is recorded in the Chinese invention patent application with the publication number CN108300688A, and will not be repeated here.
  • the resulting resuspension mixture was seeded in the first cell culture plate at a seeding density of 1 ⁇ 10 4 cells/cm 2
  • the transformation culture is performed in a cell culture incubator to obtain the first hepatic progenitor cell-like cell line.
  • the first cell culture plate is a 6-well cell culture plate, and each culture well is pre-coated with a coating solution.
  • the culture in the first cell culture plate is digested with trypsin digestion solution and then transferred to a petri dish with a diameter of 6 cm, and the first time is performed in a cell culture incubator for 7 days.
  • Expansion culture digest the culture at the bottom of a petri dish with a diameter of 6 cm with trypsin digestion solution, then transfer it to a petri dish with a diameter of 10 cm, and perform the second time in a cell culture incubator for 4 days
  • Expansion culture digest the culture at the bottom of a petri dish with a diameter of 10 cm with trypsin digestion solution, and finally transfer it to a petri dish with a diameter of 15 cm, and perform the third time in a cell culture incubator for 4 days Expansion culture.
  • the culture covering the bottom of the petri dish obtained after the third amplification culture is digested with trypsin digestion solution and then subjected to cryopreservation treatment to obtain a cryopreserved culture to be proliferated.
  • the transformation culture time is 7-14 days.
  • step S2 after thawing the frozen culture to be proliferated, it is seeded on the second cell culture plate at a seeding density of 0.8 ⁇ 10 4 cells/cm2, and the proliferation culture is carried out in a cell culture incubator for 4 days , So that the confluence rate of adherent cells obtained is 80%.
  • the confluence rate of the adherent cells is 70%-90%.
  • the second cell culture plate is a 6-well cell culture plate, and is pre-coated with a coating solution. Three days before the start of the proliferation culture, the liquid in the second cell culture plate is replaced with new TEM medium every day to achieve stable proliferation.
  • the obtained digested culture is inoculated on the third cell culture plate at a seeding density of 3 ⁇ 10 4 cells/cm 2 to Carry out the first passage for 24 hours in the cell culture incubator; after the first passage is finished, remove the supernatant in the third cell culture plate, and then add DMEM to the third cell culture plate /12 medium, lentivirus suspension and virus infection enhancement suspension to carry out the infection process in the cell culture incubator for 8 hours; after the infection process is over, replace the third medium with new DMEM/12 medium The liquid in the cell culture plate is then cultured in the cell culture incubator for 48 hours to complete the virus infection.
  • the third cell culture plate is a 6-well cell culture plate
  • the slow virus in the lentivirus suspension is a lentivirus expressing the HPVE6E7 gene
  • the virus infection enhancing suspension is a polybrene suspension.
  • the ratio of the number of lentivirus in the lentivirus suspension to the adherent cells in the third cell culture plate is 20, and 1 ml of DMEM/12 medium is added to each culture well of the third cell culture plate , 50 microliters of lentivirus and 10 microliters of the polybrene suspension, the concentration of the polybrene suspension is 8 mg/ml.
  • the ratio of the number of the lentivirus to the adherent cells in the third cell culture plate is 10-60. Specifically, the ratio of the number of the lentivirus to the number of adherent cells in the third cell culture plate is any one of 30, 40 or 50.
  • the infection process takes 6-12 hours, specifically any one of 7, 9, 10, or 11.
  • the time for continuing the culture in the cell culture incubator is 24-72 hours, specifically 36 or 60 hours.
  • the obtained digested culture is inoculated on the fourth cell culture plate at a seeding density of 3 ⁇ 10 4 cells/cm2, A second subculture with a DMEM/12 medium in a cell culture incubator for 7 days and a number of subcultures of 2.
  • the fourth cell culture plate is a 12-well cell culture plate, and is coated with a coating solution in advance.
  • the number of passages for the second subculture is 3.
  • the fourth cell culture plate is replaced with new DMEM/12 medium every 3 days. liquid.
  • the TEM medium is added to the fourth cell culture plate for the continuous selection and culture, and the resulting culture is used after the cells reach 80-90% confluence.
  • the inoculation density of 3 ⁇ 10 4 cells/cm 2 was inoculated on the fifth cell culture plate, and the continuous subculture was carried out in the cell incubator with a passage ratio of 1:3 and a number of passages of 10, and the classification was named 81.5.
  • the fifth cell culture plate is a 6-well cell culture plate, and has been coated with a coating solution in advance.
  • the process of performing the continuous subculture once is specifically as follows: observing that the cell culture to be subcultured grows to the bottom of 80-90% of the culture wells, dividing the cell culture to be subcultured into 3 parts, and substituting them into the
  • the fourth cell culture plate has the same structure in the other three cell culture plates, and 2 ml of TEM medium is added to each culture well.
  • the passage ratio of the continuous subculture is 1:2-1:4, and the number of passages is 5-10.
  • This embodiment provides a method for constructing a second immortal hepatic progenitor cell-like cell line and a second hepatic progenitor cell-like cell line obtained by the method for constructing the second immortal hepatic progenitor cell-like cell line.
  • the second human primary hepatocyte culture obtained is transformed and cultured for 14 days to obtain the second liver Progenitor-like cell line.
  • the seeding density of the second human primary hepatocyte culture is 5 ⁇ 10 4 cells/cm2.
  • the number of cultures of the expansion culture is 2, specifically the first expansion culture and the second expansion culture.
  • This example provides a method for constructing a third immortal hepatic progenitor cell-like cell line and a third hepatic progenitor cell-like cell line obtained by the method for constructing the third immortal hepatic progenitor cell-like cell line.
  • the difference between the method for constructing the third immortal hepatic progenitor cell-like cell line and the method for constructing the first immortal hepatic progenitor cell-like cell line of Example 1 is that:
  • the obtained third human primary hepatocyte culture is subjected to the transformation culture to obtain the third hepatic progenitor Cell-like cell line.
  • step S2 after thawing the frozen culture to be proliferated, it is inoculated at an inoculation density of 0.5 ⁇ 10 4 cells/cm 2.
  • This example provides a method for constructing a fourth immortal hepatic progenitor cell-like cell line and the fourth hepatic progenitor cell-like cell line obtained by the method for constructing a fourth immortal hepatic progenitor cell-like cell line.
  • the difference between the method for constructing the fourth immortal hepatic progenitor cell-like cell line and the method for constructing the first immortal hepatic progenitor cell-like cell line of Example 1 is that:
  • the obtained fourth human primary hepatocyte culture is transformed and cultured for 10 days to obtain the fourth liver Progenitor-like cell line.
  • the seeding density of the digested culture inoculated on the third cell culture plate is 2 ⁇ 10 4 cells/cm 2.
  • the infection process is 12 hours, and after the infection process is over, the culture is continued for 72 hours to complete the virus infection.
  • the inoculation density of the digested cultures inoculated on the fourth cell culture plate is 2 ⁇ 10 4 cells/cm 2.
  • the seeding density of the culture inoculated on the fifth cell culture plate is 2 ⁇ 10 4 cells/cm 2
  • the passage ratio of the continuous passage is 1:2
  • the number of passages is 5.
  • This embodiment provides a method for constructing a fifth immortal liver progenitor cell-like cell line and the fifth hepatic progenitor cell line obtained by the method for constructing the fifth immortal hepatic progenitor cell-like cell line.
  • the difference between the method for constructing the fifth immortal hepatic progenitor cell-like cell line and the method for constructing the first immortal hepatic progenitor cell-like cell line of Example 1 is that:
  • the obtained fifth human primary hepatocyte culture is subjected to the transformation culture.
  • the inoculation density of the digested cultures inoculated on the fourth cell culture plate is 4 ⁇ 10 4 cells/cm 2.
  • the seeding density of the culture inoculated on the fifth cell culture plate is 4 ⁇ 10 4 cells/cm 2
  • the passage ratio of the continuous passage is 1:4
  • the number of passages is 8.
  • This embodiment provides a method for constructing a sixth immortalized hepatic progenitor cell-like cell line and a sixth hepatic progenitor cell-like cell line obtained by the method for constructing the sixth immortalized hepatic progenitor cell-like cell line.
  • the difference between the method for constructing the sixth immortalized hepatic progenitor cell line and the method for constructing the first immortalized hepatic progenitor cell line of Example 1 is that:
  • the obtained sixth human primary hepatocyte culture is subjected to the transformation culture.
  • This example provides a heterogeneous hepatic progenitor cell-like cell bank, a hepatic progenitor cell-like cell bank, and an in vitro three-dimensional hepatocyte model.
  • the hepatic progenitor cell bank is a heterogeneous immortalized hepatic progenitor cell bank.
  • the obtained first hepatic progenitor-like cell line, the second hepatic progenitor-like cell line, the third hepatic progenitor-like cell line, and the fourth hepatic The progenitor cell-like cell line, the fifth hepatic progenitor cell-like cell line, and the sixth hepatic progenitor cell-like cell line are respectively subjected to the cryopreservation process to obtain the heterogeneous hepatic progenitor cell-like cell bank.
  • the hepatic progenitor cell-like cell bank consists of the first immortal hepatic progenitor cell-like cell line, the second immortal hepatic progenitor cell-like cell line, the third immortal hepatic progenitor cell-like cell line, and the fourth immortal hepatic progenitor cell line.
  • the hepatic progenitor cell line, the fifth immortal hepatic progenitor cell line and the sixth immortal hepatic progenitor cell line are composed.
  • the in vitro three-dimensional hepatocyte model is established using the hepatic progenitor-like cell bank.
  • the method for constructing the in vitro three-dimensional hepatocyte model was documented in the "Expansion and differentiation of human hepatocyte-derived liver progenitor-like cells for the study of hepatotropic pathogens" published in Cell Research, Volume 29, Issue 1 in 2018 In, not repeat them here.
  • the fourth immortal hepatic progenitor cell-like cell line, the fifth immortal hepatic progenitor cell-like cell line and the sixth immortal hepatic progenitor cell-like cell line obtain the first hepatocyte model strain and the second hepatocyte model, respectively Strains, the third hepatocyte model strain, the fourth hepatocyte model strain, the fifth hepatocyte model strain and the sixth hepatocyte model strain.
  • the morphological observation of the first human primary hepatocyte culture and the first hepatic progenitor-like cell line obtained after the transformation and culture was performed under a cell inverted microscope at a magnification of 40 times.
  • a schematic diagram of the morphology of human primary hepatocytes as shown in FIG. 1 and a schematic diagram of the structure of the first hepatic progenitor cell line as shown in FIG. 2 are obtained.
  • the human primary hepatocytes in the first human primary hepatocyte culture are gradually transformed into the first hepatic progenitor cell line with a more regular morphology , Is conducive to obtain stable proliferation ability.
  • the embodiment of the present invention compares the first immortal hepatic progenitor cell-like cell line, the second immortal hepatic progenitor cell-like cell line, the third immortal hepatic progenitor cell-like cell line, and the fourth immortal hepatic progenitor cell line
  • the cell lines were cultured in vitro for 40 generations, 30 generations, 20 generations, and 10 generations respectively to obtain a comparison chart of proliferation performance as shown in Figure 3 and count the corresponding doubling times.
  • the first immortal hepatic progenitor cell line, the second immortal hepatic progenitor cell line, the third immortal hepatic progenitor cell line, and The number of cells and the proliferation rate of the fourth immortal hepatic progenitor cell line are not much different, while the first immortal hepatic progenitor cell line, the second immortal hepatic progenitor cell line, the The corresponding doubling times of the third immortal hepatic progenitor cell line and the fourth immortal hepatic progenitor cell line were 29.65 ⁇ 0.4 hours, 28.32 ⁇ 0.1 hours, 30.90 ⁇ 0.3 hours and 28.92 ⁇ 0.1 hours, which can be seen by The human primary hepatocyte cultures provided by different donors in Examples 1-4, after the transformation and culture and then the cryopreservation treatment, can be successfully established for immortalization, and all have good in vitro proliferation ability.
  • the first immortal hepatic progenitor cell-like cell line, the second immortal hepatic progenitor cell-like cell line, and the first immortal hepatic progenitor cell-like cell line that were proliferated and cultured in vitro under a cell inverted microscope were respectively measured at a magnification of 100 times.
  • the three-immortal hepatic progenitor cell-like cell line and the fourth immortal hepatic progenitor-like cell line were morphologically observed, and corresponding morphological schematic diagrams as shown in FIGS. 4a to 4d were obtained.
  • the first immortal hepatic progenitor cell-like cell line has a similar morphology, and its growth characteristics are basically not affected.
  • the morphological observation of the first hepatocyte model strain in the in vitro three-dimensional hepatocyte model was performed under a cell inverted microscope at a magnification of 100 times, and the morphological schematic diagram as shown in FIG. 5 was obtained.
  • the first hepatocyte model strain has a relatively regular spherical structure, indicating good hepatocyte function.
  • the specific toxic drug Erlotinib was used to characterize the specific hepatotoxicity of the three-dimensional hepatocyte model in vitro.
  • DMSO dimethyl sulfoxide
  • Each hepatocyte model strain in the in vitro three-dimensional hepatocyte model was co-cultured with different concentrations of Erlotinib aqueous solution for 48 hours.
  • the PrestoBlue TM cell viability detection reagent produced by Life Technologies is used to separately characterize the specific hepatotoxicity of Erlotinib in each culture obtained after the co-cultivation.
  • the specific methods for the co-cultivation and the characterization of the Erlotinib-specific hepatotoxicity are routine methods used by those skilled in the art, and will not be repeated here.
  • 6a to 6f are the first hepatocyte model strain, the second hepatocyte model strain, the third hepatocyte model strain, the fourth hepatocyte model strain, and the fifth hepatocyte model Schematic diagram of Erlotinib specific hepatotoxicity of the strain and the sixth hepatocyte model strain.
  • the first hepatocyte model strain, the second hepatocyte model strain, the fifth hepatocyte model strain, and the sixth hepatocyte model strain increase with the concentration of Erlotinib solution
  • the third hepatocyte model strain and the fourth hepatocyte model strain basically have a significant downward trend with the increase in the concentration of Erlotinib solution
  • Example 8 of the present invention also provides the application of the immortal liver progenitor cell-like cell line in bioartificial liver.
  • the immortal hepatic progenitor cell line with the deposit number CCTCC NO: C2019125 is used for the application.
  • the application includes: cell proliferation, construction of animal models, establishment of extracorporeal circulation system, and sampling and testing.
  • the cell proliferation includes: loading the immortal hepatic progenitor cell strain with the preservation number of CCTCC NO: C2019125 on a carrier, and then performing cell culture through a liquid-gas interactive bioreactor to achieve hepatocyte proliferation.
  • Fig. 7 is a schematic structural diagram of a liquid-gas interactive bioreactor according to Example 8 of the present invention.
  • the liquid-gas interactive bioreactor 7 has a container body 71 and a bellows 73 that communicate with each other inside, and a liquid outlet tube 72 and a liquid inlet tube 74 disposed on the container body 71.
  • the cell culture solution used in the contents disclosed on pages 8 to 22 of the issue is introduced into the bellows 73 through the liquid inlet pipe 74, and the bellows 73 is driven to move up and down to drive the cell culture solution periodically
  • the load slide is immersed for liquid phase material exchange, and the load slide can perform gas phase material exchange with oxygen in the air in the container body 71 to perform cell proliferation.
  • the mass of the blank slide was 11 grams.
  • the rate of lifting movement of the bellows 73 is controlled to be 0.5-5 mm/s, and the time for the load carrier to perform liquid phase material exchange in each lifting cycle of the bellows 73 is 100-300 seconds, the time for gas phase material exchange is 5-120 seconds.
  • the time of the liquid phase material exchange refers to the time that the load slide is immersed in the cell culture solution, and the time of the gas phase material exchange refers to the time that the load slide is exposed to the air.
  • the cell proliferation time was 14 days.
  • the cell culture medium is replaced every 24 hours to facilitate effective three-dimensional expansion of cells.
  • the number of hepatocytes loaded on the slide was expanded from 0.5 ⁇ 10 9 cells before culture to 2.5 ⁇ 10 9 cells.
  • Cells were observed under an inverted microscope at a magnification of 100 times to obtain the morphological schematic diagram shown in FIG. 8. Referring to Figure 8, it can be seen that the obtained proliferating cells have a relatively regular morphological structure, which is conducive to exerting good cell functions.
  • Example 8 of the present invention the gene expression levels of different functional genes of proliferating cells obtained at different proliferation times were further investigated to investigate cell functions.
  • the RNAfast200 kit with the article number 220010 produced by Shanghai Feijie Biotechnology Co., Ltd. was used to extract the RNA of the proliferating cells, and then the reverse transcription with the article number 18064014 produced by Invitrogen was used.
  • the enzyme reverse-transcribes the obtained RNA into cDNA, and finally expresses the relevant functional genes separately by fluorescent quantitative PCR.
  • the specific expression method of the fluorescent quantitative PCR is a conventional method of those skilled in the art, and will not be repeated here.
  • CPS1 carbamoyl phosphate synthetase 1
  • ⁇ 1-antitrypsin Alpha-1-antitrypsin, AAT
  • albumin Albumin, Alb
  • drug metabolism enzyme CYP3A4 Protein coding gene GSTA2 and multidrug resistance related protein MRP4.
  • Table 1 for the average gene expression levels of CPS1, AAT, Alb, CYP3A4, GSTA2 and MRP4 of the proliferating cells obtained after 2 days of cell proliferation and the proliferating cells obtained after 14 days of cell proliferation.
  • the construction of the animal model includes: dissolving D-(+)-Galactosamine hydrochloride (D-gal) in an aqueous glucose solution with a mass concentration of 5%, dilute to 0.5g/mL and then use 1mol/mL.
  • the pH value of L sodium hydroxide aqueous solution is adjusted to 6.8 to form a D-gal solution; the D-gal solution is filtered and sterilized using a syringe filter with an average pore size of 0.22 microns.
  • Fig. 9 is a schematic structural diagram of an extracorporeal circulation system of embodiment 8 of the present invention.
  • the establishment of the extracorporeal circulation system includes:
  • the blood of the experimental piglet is connected to the first catheter 91 through the femoral vein puncture catheter, and is pumped to the plasma separator 92 while passing through the first catheter 91
  • Add heparin to the plasma separator 92, and the total heparin dose is controlled to 100 units per kilogram of piglets.
  • the plasma separator 92 pumps the separated plasma into the liquid-gas interactive bioreactor 7 through the second conduit 93, and the liquid-gas interactive bioreactor 7 is placed after the cell proliferation
  • the obtained slide is then controlled by the liquid-gas interactive bioreactor 7 to control the liquid-phase material exchange and the gas-phase material exchange between the plasma and the slide.
  • the lifting of the bellows 73 is controlled
  • the speed of movement is 2 mm/sec.
  • the time for the load carrier to perform liquid phase material exchange is 120 seconds, and the time for gas phase material exchange to be 10 seconds.
  • the specific control method please refer to the description of the cell proliferation, which will not be repeated here.
  • the obtained detoxified plasma is pumped into the plasma separator 92 through the third conduit 94 to be remixed with blood cells to form purified blood, and returned to the experimental piglets through the fourth conduit 95 to complete the circulatory treatment process.
  • the total time of the cycle treatment is 3h.
  • the sampling and testing includes: taking blood from the experimental piglets at different times within 72 hours after the end of the cycle treatment, and inspecting serum biochemical indicators and liver coagulation ability indicators.
  • the embodiment of the present invention provides comparative example 1.
  • the blood of experimental piglets is filtered through a bilirubin adsorption column of model BS330 and a hollow fiber tube, it flows through the blank of the blood through the extracorporeal circulation system shown in FIG. Liquid-gas interactive bioreactor 7 for 3 hours cycle treatment.
  • the blank slide in the liquid-gas interactive bioreactor 7 is a blank slide, and the rest of the specific control process please refer to the foregoing.
  • the serum biochemical indexes of Example 8 and Comparative Example 1 are ALT content, AST content, serum ammonia (Ammonia) content, total bilirubin (TBiL) content, and lactate dehydrogenase (LDH) content And Alb content, see Table 2 for specific values.
  • the unit of ALT and AST is U/L
  • the unit of Ammonia, TBiL and LDH is ⁇ mol/L
  • the unit of Alb is g/L.
  • the ALT of Comparative Example 1 increased sharply from 200 U/L to 2500 U/L 24 hours after the end of the cycle treatment. , And then dropped sharply to 700 U/L, while the ALT of Example 8 always remained within 200 U/L, and there was a slow downward trend within 48 to 72 hours after the end of the cycle treatment.
  • the Ammonia of Comparative Example 1 and Example 8 increased to 80 ⁇ mol/L and 50 ⁇ mol/L, respectively, within 24 hours after the end of the cycle treatment. Then, the Ammonia of Example 8 continued to decrease until it was not higher than 10 ⁇ mol/L, and The Ammonia of Comparative Example 1 significantly increased to 450 ⁇ mol/L after 24 hours of slow decrease.
  • the TBiL of Comparative Example 1 significantly increased to 60 ⁇ mol/L within 48 hours after the end of the cycle treatment, and then dropped again To 56.7 ⁇ mol/L.
  • the TBiL of Example 8 never exceeded 3.5 ⁇ mol/L.
  • Example 8 From the data of TBiL, it can be seen that after detoxification of the proliferating cells formed by the proliferation of the immortal hepatic progenitor-like cell line in Example 8, the experimental piglets of Example 8 showed better liver secretion and excretion functions than those of Comparative Example 1. Experimental piglet.
  • Example 8 72 hours after the end of the cycle treatment, for Example 8, the amplitude of Alb decrease was very low. From the data measured at different times within 72 hours after the end of the cycle treatment, the implementation The Alb of Example 8 varies between 36-40g/L.
  • the Alb of Comparative Example 1 decreased more than that in Example 8. From the data measured at different times within 72 hours after the end of the cycle treatment, The Alb of Comparative Example 1 varied between 29-35g/L.
  • the LDH of Comparative Example 1 was much higher than that of Example 8, showing abnormal activity, measured at different times within 72 hours after the end of the cycle treatment From the data point of view, the LDH of Comparative Example 1 has maintained a significant upward trend since the end of the cycle treatment. Although the LDH of Example 8 also has an upward trend, the increase rate is much lower than that of the Comparative Example.
  • liver coagulation ability indicators include prothrombin time (PT), thrombin time (TT), activated partial thromboplastin time (APTT), and INR.
  • PT prothrombin time
  • TT thrombin time
  • APTT activated partial thromboplastin time
  • INR INR
  • the PT of Comparative Example 1 significantly increased from 20 seconds to 150 seconds from the 24th hour to the 72th hour after the end of the cyclic treatment, while the PT of Example 8 never exceeded 30 seconds, and since the end of the cyclic treatment From the 24th to the 72nd hour, PT had a slow downward trend, and finally dropped to 12 seconds.
  • the INR of Comparative Example 1 increased significantly from 1.78 to 8 from the 24th hour to the 72nd hour after the end of the cycle treatment, while the INR of Example 8 never exceeded 2.6, and from the 24th hour to the 72nd hour after the end of the cycle treatment At the 72nd hour, APTT had a slow downward trend, and finally dropped to 1.05.
  • the experimental piglet of Comparative Example 1 and the experimental piglet of Example 8 showed little difference in TT measured within 48 hours after the end of the cycle treatment, and both showed an increasing trend, specifically from about 20 seconds to 40 seconds Around 72 hours after the end of the cycle treatment, the TT of Example 8 dropped to 21 seconds, while the TT of Comparative Example 1 continued to rise to about 50 seconds.
  • the APTT measured by the experimental piglet of Comparative Example 1 and the experimental piglet of Example 8 showed a slow increase trend within 24 hours after the end of the cycle treatment. Specifically, the APTT of Comparative Example 1 increased to 20 seconds. The APTT of 8 increased to 28 seconds; within 24 hours to 72 hours after the end of the cycle treatment, the APTT of Comparative Example 1 significantly increased to 60 seconds, while the APTT of Example 8 had a downward trend, and finally dropped to 18 seconds.
  • liver coagulation ability Based on the above indicators of liver coagulation ability, it can be seen that the liver of the experimental piglet of Comparative Example 1 has a significantly reduced ability to synthesize coagulation factors, and the degree of liver damage is high.
  • the use of the proliferating cells formed by the proliferation of the immortal hepatic progenitor-like cell line of Example 8 to detoxify plasma has a positive auxiliary effect on the normalization of the aforementioned serum biochemical indicators and liver coagulation ability indicators.
  • the liquid-gas interactive bioreactor 7 is perfused with the plasma of the experimental piglets
  • the plasma of the experimental piglets and the proliferating cells formed by the proliferation of immortal hepatic progenitor-like cell lines pass through the aforementioned gaseous substances
  • at least one exogenous human growth factor was detected in the purified plasma obtained, thereby proving that the cells formed by the proliferation of the immortal hepatic progenitor-like cell line of the embodiment of the present invention are used in biology
  • the artificial liver can secrete at least one exogenous human growth factor during treatment.
  • the at least one exogenous human growth factor includes human hepatocyte growth factor (Human HGF) and human transforming growth factor- ⁇ (Human Transforming Growth Factor- ⁇ , Human TGF- ⁇ ).
  • Human HGF human hepatocyte growth factor
  • human transforming growth factor- ⁇ Human Transforming Growth Factor- ⁇ , Human TGF- ⁇
  • ⁇ Any one or more of human interleukin-6 (Human Interleukin-6, Human IL-6).
  • Example 8 and Comparative Example 1 sampled and analyzed Human HGF, Human TGF- ⁇ , Human from the plasma of the liquid-gas interactive bioreactor 7 at different times. Please refer to Table 3 for the values of IL-6, urea and lactic acid content.
  • the at least one exogenous human growth factor is Human HGF, Human TGF- ⁇ , and Human IL-6, which help to play a positive role in promoting early liver recovery .
  • the urea level in the purified plasma increased, indicating that the cells formed by the proliferation of the immortal hepatic progenitor-like cell line in the embodiment of the present invention are helpful in detoxifying the ammonia-based toxic substances in the experimental piglet plasma.
  • the decrease in the concentration of lactic acid in the purified plasma further proves that the cells formed by the proliferation of the immortal hepatic progenitor-like cell line of the embodiment of the present invention have liver metabolism function.
  • Example 9 of the present invention provides the application of the immortalized hepatic progenitor cell-like cell line in liver cell transplantation, including the construction of liver failure models in mice, the preparation and transplantation of in vivo bioreactors.
  • the construction of the mouse acute liver failure model includes: providing olive oil containing carbon tetrachloride as an inducer, and subcutaneously injecting 200 microliters of 10% carbon tetrachloride into experimental mice with severe immunodeficiency in NSG. On the next day, it is advisable to take a blood sample from the orbital vein as a control to lose 1-3 g, and the state is slightly worse than the previous day, so as to complete the construction of the mouse model.
  • the preparation of the in vivo bioreactor includes: preparing cell pellets from the sodium alginate aqueous solution and the suspension cell mass formed by the proliferating cells prepared from the immortalized hepatic progenitor cell line with the deposit number CCTCC NO: C2019125 in Example 8. Microcapsules, and used as the in vivo bioreactor.
  • the specific preparation method includes: providing a sodium alginate aqueous solution with a mass and volume concentration of 1.5% prepared in sterile physiological saline; uniformly mixing the sodium alginate aqueous solution with the suspended cell mass, so that each milliliter of the mixture formed Containing 3 ⁇ 10 6 cells; use an IE-50R packaging machine equipped with a D250 micron sterile nozzle to spray the mixed solution into a 1.2% calcium chloride aqueous solution for cross-linking for 10 minutes to obtain wet Cell ball microcapsules; the wet cell ball microcapsules are washed with physiological saline to remove free calcium ions and then washed with deionized water to obtain the cell ball microcapsules.
  • the average diameter of the cell balloon microcapsules after washing with deionized water does not exceed 500 microns.
  • Fig. 10 is an electron micrograph of a cell ball microcapsule according to some embodiments of the present invention.
  • sodium alginate has good biocompatibility and pH sensitivity, it can quickly form a hydrogel under mild conditions, and bind biologically active molecules through salt bonds, hydrogen bonds or hydrophobic interactions.
  • the inside of the capsule 101 is coated with the proliferating cell cluster 102, forming the in vivo bioreactor.
  • the diameter of the in vivo bioreactor is approximately 85 microns.
  • the transplantation of the in vivo bioreactor includes: redispersing the in vivo bioreactor with physiological saline, and then transplanting the mouse model into the abdominal cavity.
  • the embodiment of the present invention also provides a comparative example 2, using dispersed microcapsules without proliferating cells to transplant the mouse model into the abdominal cavity.
  • the difference between the method for preparing microcapsules without proliferating cells and the method for preparing cell ball microcapsules is that the suspended cell cluster is not added to the sodium alginate aqueous solution.
  • Example 9 Seven days after the completion of the abdominal cavity transplantation, the surviving experimental mice of Example 9 were euthanized and then dissected and observed. It was found that the internal bioreactors were scattered throughout the abdominal cavity and loosely connected to the mesentery and omentum.
  • the results of the survival analysis showed that the survival rate of Example 9 dropped to 70% on the 2nd day after the end of the intraabdominal transplantation; as the observation time was extended to the 7th day after the end of the intraabdominal transplantation, implementation The survival rate of Example 9 remained unchanged at 70%, and the health status was rated as semi-dependent; the survival rate of Comparative Example 2 decreased by 15-20% every day. On the 7th day after the end of the abdominal transplantation, the survival rate of Comparative Example 2 Decrease to 15%, and the health status is assessed as seriously ill.
  • Example 9 and Comparative Example 2 were tested for serum ALT and AST after the end of the abdominal cavity transplantation and on the 1, 3, and 7 days after the end of the abdominal cavity transplantation. The results showed that on the first day after the end of the abdominal cavity transplantation, The serum ALT and AST of Example 9 both decreased by 70-80%, while the serum ALT and AST of Comparative Example 2 were both decreased by 30-40%. On the 3-7 days after the abdominal cavity transplantation, Example 9 and Comparative Example The serum ALT and AST of 2 surviving mice dropped to normal levels.
  • the proliferating cells prepared by the immortal hepatic progenitor-like cell line can help alleviate the degree of liver damage in the mouse model.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • General Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Virology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Pathology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Physics & Mathematics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Physiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)

Abstract

提供了一种肝祖细胞样细胞库的构建方法，包括：对不同供体来源的人原代肝细胞培养物依次进行转化培养、冻存处理、增殖培养、第一传代处理、病毒感染、第二传代处理、连续筛选培养以及连续传代培养。所述异质性永生肝祖细胞样细胞库构建方法中，对每个供体来源的人原代肝细胞培养物在进行所述增殖培养前先进行转化培养，有利于赋予所述人原代肝细胞培养物良好的增殖性能，结合后续的培养参数控制，使得到的不同供体来源的永生肝祖细胞样细胞株具有良好的体外增殖能力。还提供了所述肝祖细胞样细胞库的应用以及通过所述构建方法得到的细胞株。

Description

肝祖细胞样细胞库的构建方法及其制备的细胞株与应用 技术领域
本发明涉及生物技术领域，尤其涉及肝祖细胞样细胞库的构建方法及其制备的细胞株与应用。
背景技术
肝衰竭是严重肝病的终末期表现，患者病死率为可高达50％～90％。其中，患者因药物不良反应导致药物性肝损伤的问题较突出，占急性肝衰竭病例的50％以上，从而导致药物开发失败或上市后撤回。
药物开发失败或上市后撤回的大部分原因是由于存在对患者造成特异性毒性。现有技术中，肝细胞永生化是解决肝细胞来源的重要途径之一，通常对原代肝细胞或诱导干细胞分化而来的肝细胞样细胞进行永生化处理，将得到的细胞株应用于肝脏代谢异质性研究，以寻求对药物特异性毒性问题的解决之道。
公开号为CN108330099A的中国发明专利申请公开了一种个性化肝细胞的培养和扩增方法，该方法将经过基因修饰的肝实质细胞置入肝细胞增殖培养基中进行培养和扩增，再对获得的肝细胞进行分化培养，得到了成熟肝实质细胞。然而，该方法先对肝实质细胞进行基因修饰，再进行后续的增殖培养和分化培养，容易影响得到的成熟肝实质细胞的体外增殖能力，且供体来源单一，无法开展药物特异性毒性研究。
因此有必要开发一种新型的肝祖细胞样细胞库构建方法及应用以避免现有技术中存在的上述问题。
发明内容
本发明的目的在于提供肝祖细胞样细胞库的构建方法及其制备的细胞株与应用，以获得具有良好体外增殖能力的永生肝祖细胞样细胞株，并有利于开展对药物特异性毒性的研究。
为实现上述目的，本发明的所述肝祖细胞样细胞库的构建方法包括：
S1：提供不同供体来源的人原代肝细胞培养物，对每个供体来源的人原代肝细胞培养物在0.5×10 4-5×10 4个/平方厘米的接种密度下进行7-14天的转化培养，然后对经所述转化培养后得到的每个供体来源的肝祖细胞样细胞系进行冻存处理，以得到异质性肝祖细胞样细胞库；
S2：对每个供体来源的肝祖细胞样细胞系解冻后进行增殖培养，以分别得到不同供体来源的贴壁细胞，每个供体来源的贴壁细胞的汇合率为70％-90％；
S3：对每个供体来源的贴壁细胞依次进行第一传代处理和病毒感染，并在所述病毒感染的过程中进行培养基置换；
S4：对经所述病毒感染后得到的每个供体来源的培养物进行传代次数为2或3的第二传代处理，通过筛选培养基对经所述第二传代处理后得到的培养物进行连续筛选培养，以得到不同供体来源的感染培养物；
S5：对每个供体来源的感染培养物进行传代比例为1:2-1:4，传代次数为5-10的连续传代培养，以得到所述肝祖细胞样细胞库。
本发明的所述肝祖细胞样细胞库构建方法的有益效果在于：在所述步骤S11中，对每个供体来源的人原代肝细胞培养物在0.5×10 4-5×10 4个/平方厘米的接种密度下进行7-14天的转化培养，有利于赋予所述人原代肝细胞培养物良好的增殖性能，结合后续对所述第二传代处理、所述筛选处理以及所述连续传代培养的控制，继而使得到的不同供体来源的永生肝祖细胞样细胞株均具有良好的体外增殖能力，从而有利于开展对药物特异性毒性的研究。
优选的，所述步骤S1中，所述转化培养结束后，对经所述转化培养后得到的培养物依次进行2或3次的扩增培养后，再执行所述冻存处理。其有益效果 在于：进一步赋予所述人原代肝细胞培养物良好的增殖性能。
优选的，所述步骤S2中，所述肝祖细胞样细胞系在0.5×10 4-5×10 4个/平方厘米的接种密度下进行所述增殖培养，所述增殖培养开始后的三天内，每天进行培养基置换。其有益效果在于：能够在保证细胞活性的同时尽快得到贴壁细胞。
优选的，所述步骤S3中，所述每个供体来源的贴壁细胞在2×10 4-4×10 4个/平方厘米的接种密度下进行24小时的所述第一传代处理。其有益效果在于：合适的接种密度有利于后续的病毒感染有效进行。
优选的，所述步骤S3中，经所述第一传代处理后得到的培养物在2×10 4-4×10 4个/平方厘米的接种密度下进行所述病毒感染。其有益效果在于：合适的接种密度有利于病毒感染有效进行。
进一步优选的，向经所述第一传代处理后得到的培养物中加入DMEM/12培养基、慢病毒和聚凝胺悬液后的6-12小时后进行培养基置换，然后继续培养24-72小时，以完成所述病毒感染，所述慢病毒与所述贴壁细胞的数量比为0.5-60。
优选的，所述步骤S4中，经所述病毒感染后得到的每个供体来源的培养物在2×10 4-4×10 4个/平方厘米的接种密度下进行5-7天的所述第二传代处理，所述第二传代处理的过程中，每2-3天进行一次培养基置换。
优选的，所述筛选培养基为TEM培养基。
优选的，所述步骤S5中，所述每个供体来源的感染培养物以2×10 4-4×10 4个/平方厘米的接种密度进行所述连续传代培养。
由于通过所述构建方法制备的所述异质性永生肝祖细胞样细胞库中的永生肝祖细胞样细胞株具有良好体外增殖能力，通过所述异质性永生肝祖细胞样细胞库构建体外三维肝细胞模型，然后利用所述体外三维肝细胞模型检测药物的特异质肝毒性，有利于开展对药物特异性毒性的研究。另外，通过所述构建方法制备的永生肝祖细胞样细胞株能够应用于生物人工肝和肝细胞移植。
本发明还提供了利用所述构建方法制备的分类命名为81.5的永生肝祖细胞样细胞株，所述永生肝祖细胞样细胞株保藏于位于中国武汉的武汉大学内的中国典型培养物保藏中心，保藏编号为CCTCC NO:C2019125。 具体的，中国典型培养物保藏中心于2019年6月11日收到培养物并命名为永生化人肝祖细胞样细胞系ALI-CELL-81.5, 且于2019年6月24日检测认定该培养物为存活状态。所述永生化人肝祖细胞样细胞系ALI-CELL-81.5 即本发明所述的分类命名为81.5的永生肝祖细胞样细胞株。 中国典型培养物保藏中心应请求由2019年6月U日起对培养物保存30年，并在期满前收到提供培养物样品的请求后再延续保存5年。
本发明还提供了所述永生肝祖细胞样细胞株在生物人工肝和肝细胞移植方面的应用，所述永生肝祖细胞样细胞株增殖形成的肝细胞应用于生物人工肝治疗的过程中分泌外源性人类生长因子。
优选的，所述至少一种外源性人类生长因子包括人源的肝细胞生长因子、人源的转化生长因子-α和人源的白介素-6中的任意一种或多种。
附图说明
图1为本发明的人原代肝细胞的形态示意图；
图2为本发明的第一肝祖细胞样细胞系的形态示意图；
图3为本发明的第一永生肝祖细胞样细胞株、第二永生肝祖细胞样细胞株、第三永生肝祖细胞样细胞株以及第四永生肝祖细胞样细胞株的增殖性能对比图；
图4a为本发明的经体外增殖培养后的第一永生肝祖细胞样细胞株的形态示意图；
图4b为本发明的经体外增殖培养后的第二永生肝祖细胞样细胞株的形态示意图；
图4c为本发明的经体外增殖培养后的第三永生肝祖细胞样细胞株的形态示意图；
图4d为本发明的经体外增殖培养后的第四永生肝祖细胞样细胞株的形态示意图；
图5为本发明的第一肝细胞模型株的形态示意图；
图6a本发明的第一肝细胞模型株的Erlotinib特异质肝毒性示意图；
图6b为本发明的第二肝细胞模型株的Erlotinib特异质肝毒性示意图；
图6c为本发明的第三肝细胞模型株的Erlotinib特异质肝毒性示意图；
图6d为本发明的第四肝细胞模型株的Erlotinib特异质肝毒性示意图；
图6e为本发明的第五肝细胞模型株的Erlotinib特异质肝毒性示意图；
图6f为本发明的第六肝细胞模型株的Erlotinib特异质肝毒性示意图；
图7为本发明的液气交互式生物反应器的结构示意图；
图8为本发明经细胞增殖后得到的增殖细胞在细胞倒置显微镜下以100倍的放大倍数进行观察得到的形态示意图；
图9为本发明的体外循环系统的结构示意图；
图10为本发明的细胞球微囊的电镜照片。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合本发明的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。除非另外定义，此处使用的技术术语或者科学术语应当为本发明所属领域内具有一般技能的人士所理解的通常意义。本文中使用的“包括”等类似的词语意指出现该词前面的元件或者物件涵盖出现在该词后面列举的元件或者物件及其等同，而不排除其他元件或者物件。
具体实施方式中的主要试剂来源如下：
TEM培养基来源于上海赛立维生物科技有限公司；基质胶产自美国康宁(Corning)公司，货号为356234；聚凝胺悬液产自上海翊圣生物科技有限公司，货号为40804ES76；胰酶消化液、DMEM/12培养基和HepX培养基产自上海源培生物科技股份有限公司，货号分别为S310KJ、L110KJ以及X071A1。
具体实施方式中的主要仪器来源如下：
不同孔数的细胞培养板以及不同直径的培养皿均产自耐思科学有限公司(NEST ScienceCo.Ltd)；细胞培养孵箱购自新加坡艺思高科技有限公司(ESCO)，产品型号为CLL-170B-8；细胞倒置显微镜购自株式会社尼康(Nikon)，产品型号为Ta2-FL。
针对现有技术存在的问题，本发明的实施例提供了一种肝祖细胞样细胞库的构建方法，包括：
S1：提供不同供体来源的人原代肝细胞培养物，对每个供体来源的人原代肝细胞培养物在0.5×10 4-5×10 4个/平方厘米的接种密度下进行7-14天的转化培养，然后对经所述转化培养后得到的每个供体来源的肝祖细胞样细胞系进行冻存处理，以得到异质性肝祖细胞样细胞库；
S2：对每个供体来源的肝祖细胞样细胞系解冻后进行增殖培养，以分别得到不同供体来源的贴壁细胞，每个供体来源的贴壁细胞的汇合率为70％-90％；
S3：对每个供体来源的贴壁细胞依次进行第一传代处理和病毒感染，并在所述病毒感染的过程中进行培养基置换；
S4：对经所述病毒感染后得到的每个供体来源的培养物进行传代次数为2或3的第二传代处理，通过筛选培养基对经所述第二传代处理后得到的培养物进行连续筛选培养，以得到不同供体来源的感染培养物；
S4：对每个供体来源的感染培养物进行传代比例为1:2-1:4，传代次数为5-10的连续传代培养，以得到所述肝祖细胞样细胞库。
本发明实施例中的所述肝祖细胞样细胞库为异质性的永生化肝祖细胞样细胞库。
本发明一些实施例中，利用细胞冻存液和液氮进行所述冻存处理。
本发明一些实施例中，进行所述病毒感染使用的病毒记载在公开号为CN108330099A的中国发明专利申请中。具体的，为表达SV40病毒大T抗原基 因、HPV病毒E6E7基因或泛素连接酶基因。
本发明一些实施例中，所述筛选培养基为TEM培养基。
本发明一些实施例的所述步骤S1中，所述转化培养结束后，对经所述转化培养后得到的培养物依次进行2-3次的扩增培养和所述冻存处理。
本发明一些实施例的所述步骤S2中，所述肝祖细胞样细胞系在0.5×10 4-5×10 4个/平方厘米的接种密度下进行所述增殖培养，所述增殖培养开始后的三天内，每天进行培养基置换。
本发明实施例中，通过所述构建方法制备的永生肝祖细胞样细胞株能够应用于生物人工肝、肝细胞移植以及药物的特异质肝毒性检测。
以下通过实施例1-7进行详细阐述，其中所述的不同供体均来源于上海东方肝胆外科医院的废弃手术标本，术前征得患者本人同意并签署知情同意书。
实施例1
本实施例提供了第一肝祖细胞样细胞系、分类命名为81.5，保藏于中国典型培养位保藏中心，保藏编号为CCTCC NO：C2019125的第一永生肝祖细胞样细胞株，以及所述第一永生肝祖细胞样细胞株的构建方法。
本实施例中的包被液为HepX培养基与基质胶的混合液，所述HepX培养基与所述基质胶的体积比为80:1。
当培养器为6孔细胞培养板，向每个培养孔中加入250-350微升的包被液以预先进行所述包被处理。
当培养器为12孔细胞培养板，向每个培养孔中加入150-200微升的包被液以预先进行所述包被处理。
当培养器为直径6厘米的细胞培养皿，加入500-700微升包被液以进行包被处理。
当培养器为直径10厘米的细胞培养皿，加入1000-1500微升包被液以进行包 被处理.
培养器为直径15厘米的细胞培养皿，加入2000-3000微升包被液以进行包被处理。
所述包被处理的时间为30-120分钟。
本实施例涉及的细胞培养孵箱，内部为37℃恒温，饱和湿度，二氧化碳的浓度为5％。所述细胞培养孵箱内进行的过程除外，其他操作均在常温无菌操作台中进行。
本实施例中，利用细胞消化液对培养物进行消化的具体实施方式为本领域技术人员的常规技术手段，在此不做赘述。所述细胞消化液为胰酶消化液。
所述第一永生肝祖细胞样细胞株的构建方法具体为：
所述步骤S1中，提供第一人原代肝细胞培养物，对所述第一人原代肝细胞培养物在1×10 4个/平方厘米的接种密度下进行7天的转化培养，然后对经所述转化培养后得到的第一肝祖细胞样细胞系依次进行3次扩增处理。
具体的，采用两步灌注法或切碎消化法中的任意一种对来源于第一供体的人肝血管瘤旁组织进行分离处理，以得到所述第一人原代肝细胞培养物，所述两步灌注法的具体实施方式记载在2018年发表在Cell Research(细胞研究)第29卷第1期的“Expansion and differentiation of human hepatocyte-derived liver progenitor-like cells for the study of hepatotropic pathogens”中，所述切碎消化法的具体实施方式记载在公开号为CN108300688A的中国发明专利申请中，在此均不做赘述。
用TEM培养基对所述第一人原代肝细胞培养物进行重悬后，将得到的重悬混合物在1×10 4个/平方厘米的接种密度接种于第一细胞培养板中，以在细胞培养孵箱内进行所述转化培养，得到第一肝祖细胞样细胞系。所述第一细胞培养板为6孔细胞培养板且每个培养孔预先用包被液进行包被处理。
所述转化培养结束后，利用胰酶消化液消化所述第一细胞培养板中的培养物后转移到直径为6厘米的培养皿中，并在细胞培养孵箱内进行7天的第一次扩增培养；利用胰酶消化液消化长满直径为6厘米的培养皿底部的培养物，然后转移至直径为10厘米的培养皿中，并在细胞培养孵箱内进行4天的第二次扩增培养；利用胰酶消化液消化长满直径为10厘米的培养皿底部的培养物，最后转移至直径为15厘米的培养皿中，并在细胞培养孵箱内进行4天的第三次扩增培养。对经所述第三次扩增培养后得到的长满培养皿底部的培养物用胰酶消化液进行消化后进行冻存处理，得到冻存待增殖培养物。
本发明一些实施例中，所述转化培养的时间为7-14天。
所述步骤S2中，解冻所述冻存待增殖培养物后，以0.8×10 4个/平方厘米的接种密度接种于第二细胞培养板，并在细胞培养孵箱内进行4天的增殖培养，以使获得的贴壁细胞的汇合率为80％。
本发明一些实施例中，所述贴壁细胞的汇合率为70％-90％。
具体的，所述第二细胞培养板为6孔细胞培养板，且预先用包被液进行了包被处理。所述增殖培养开始的前三天，每天用新的TEM培养基置换所述第二细胞培养板中的液体，以实现稳定增殖。
所述步骤S3中，利用胰酶消化液消化经所述增殖培养得到的培养物后，将得到的消化培养物以3×10 4个/平方厘米的接种密度接种于第三细胞培养板，以在细胞培养孵箱内进行24小时的第一传代处理；所述第一传代处理结束后，去除所述第三细胞培养板中的上清液，然后向所述第三细胞培养板中加入DMEM/12培养基、慢病毒悬液和病毒感染增强悬液，以在细胞培养孵箱内进行8小时的感染过程；所述感染过程结束后，用新的DMEM/12培养基置换所述第三细胞培养板内的液体，然后继续在细胞培养孵箱内进行48小时的培养，以完成所述病毒感染。
具体的，所述第三细胞培养板为6孔细胞培养板，所述慢病毒悬液中的慢病 毒为表达HPVE6E7基因的慢病毒，所述病毒感染增强悬液为聚凝胺悬液。所述慢病毒悬液中的慢病毒与所述第三细胞培养板中的贴壁细胞的数量比为20，所述第三细胞培养板的每个培养孔中加入1毫升DMEM/12培养基、50微升慢病毒和10微升所述聚凝胺悬液，所述聚凝胺悬液的浓度为8毫克/毫升。
本发明一些实施例中，所述慢病毒与所述第三细胞培养板中的贴壁细胞的数量比为10-60。具体的，所述慢病毒与所述第三细胞培养板中的贴壁细胞的数量比为30、40或50中的任意一种。
本发明一些实施例中，所述感染过程的时间为6-12小时，具体为7、9、10或11中的任意一种。所述感染过程结束后，继续在细胞培养孵箱内培养的时间为24-72小时，具体为36或60小时。
所述步骤S4中，利用胰酶消化液消化经所述病毒感染后得到的培养物后，将得到的消化培养物以3×10 4个/平方厘米的接种密度接种于第四细胞培养板，以在细胞培养孵箱内通过DMEM/12培养基进行7天，传代次数为2的第二传代培养。
具体的，所述第四细胞培养板为12孔细胞培养板，且预先用包被液进行了包被处理。
所述第二传代培养的过程中，每2天用新的DMEM/12培养基置换所述第四细胞培养板中的液体。
本发明一些实施例中，所述第二传代培养的传代次数为3，所述第二传代培养的过程中，每3天用新的DMEM/12培养基置换所述第四细胞培养板中的液体。
所述步骤S4中，所述第二传代培养结束后，向所述第四细胞培养板中加入TEM培养基进行所述连续筛选培养，细胞达到80-90％汇合率后将得到的培养物以3×10 4个/平方厘米的接种密度接种于第五细胞培养板，以在细胞孵箱内进行传代比例为1:3，传代次数为10的连续传代培养，以得到分类命名为81.5，保藏于中国典型培养位保藏中心，保藏编号为CCTCC NO：C2019125的第一永生 肝祖细胞样细胞株。
具体的，所述第五细胞培养板为6孔细胞培养板，且预先用包被液进行过包被处理。
进行一次所述连续传代培养的过程具体为：观察到待传代细胞培养物生长到80-90％培养孔的底部，将所述待传代细胞培养物平均分成3份后，分别传代入与所述第四细胞培养板结构相同的其他3个细胞培养板中，且向每个培养孔中加入2毫升TEM培养基。
本发明一些实施例中，所述连续传代培养的传代比例为1:2-1:4，传代次数为5-10。
实施例2
本实施例提供了第二永生肝祖细胞样细胞株的构建方法以及通过所述第二永生肝祖细胞样细胞株的构建方法得到的第二肝祖细胞样细胞系。
所述第二永生化肝祖细胞株的构建方法与实施例1的所述第一永生肝祖细胞样细胞株的构建方法的区别在于：
所述步骤S1中，对来源于第二供体的人肝血管瘤旁组织进行分离处理后，对得到的第二人原代肝细胞培养物进行14天的转化培养，得到所述第二肝祖细胞样细胞系。所述第二人原代肝细胞培养物的接种密度为5×10 4个/平方厘米。所述扩增培养的培养次数为2，具体为所述第一次扩增培养和所述第二次扩增培养。
实施例3
本实施例提供了第三永生肝祖细胞样细胞株的构建方法以及通过所述第三永生肝祖细胞样细胞株的构建方法得到的第三肝祖细胞样细胞系。
所述第三永生肝祖细胞样细胞株的构建方法与实施例1的所述第一永生肝祖细胞样细胞株的构建方法的区别在于：
所述步骤S1中，对来源于第三供体的人肝血管瘤旁组织进行分离处理后，对得到的第三人原代肝细胞培养物进行所述转化培养，得到所述第三肝祖细胞样细胞系。
所述步骤S2中，解冻所述冻存待增殖培养物后，以0.5×10 4个/平方厘米的接种密度接种。
实施例4
本实施例提供了第四永生肝祖细胞样细胞株的构建方法以及通过所述第四永生肝祖细胞样细胞株的构建方法得到的第四肝祖细胞样细胞系。
所述第四永生肝祖细胞样细胞株的构建方法与实施例1的所述第一永生肝祖细胞样细胞株的构建方法的区别在于：
所述步骤S1中，对来源于第四供体的人肝血管瘤旁组织进行分离处理后，对得到的第四人原代肝细胞培养物进行10天的转化培养，得到所述第四肝祖细胞样细胞系。
所述步骤S3中，接种于所述第三细胞培养板的消化培养物的接种密度为2×10 4个/平方厘米。所述感染过程为12小时，所述感染过程结束后，继续培养72小时，以完成所述病毒感染。
所述步骤S4中，接种于所述第四细胞培养板的消化培养物的接种密度为2×10 4个/平方厘米。
所述步骤S5中，接种于所述第五细胞培养板的培养物的接种密度为2×10 4个/平方厘米，所述连续传代的传代比例为1:2，传代次数为5。
实施例5
本实施例提供了第五永生肝祖细胞样细胞株的构建方法以及通过所述第五永生肝祖细胞样细胞株的构建方法得到的第五肝祖细胞样细胞系。
所述第五永生肝祖细胞样细胞株的构建方法与实施例1的所述第一永生肝祖 细胞样细胞株的构建方法的区别在于：
所述步骤S1中，对来源于第五供体的人肝血管瘤旁组织进行分离处理后，对得到的第五人原代肝细胞培养物进行所述转化培养。
所述步骤S4中，接种于所述第四细胞培养板的消化培养物的接种密度为4×10 4个/平方厘米。
所述步骤S5中，接种于所述第五细胞培养板的培养物的接种密度为4×10 4个/平方厘米，所述连续传代的传代比例为1:4，传代次数为8。
实施例6
本实施例提供了第六永生化肝祖细胞样细胞株的构建方法以及通过所述第六永生化肝祖细胞样细胞株的构建方法得到的第六肝祖细胞样细胞系。
所述第六永生化肝祖细胞样细胞株的构建方法与实施例1的所述第一永生肝祖细胞样细胞株的构建方法的区别在于：
所述步骤S1中，对来源于第六供体的人肝血管瘤旁组织进行分离处理后，对得到的第六人原代肝细胞培养物进行所述转化培养。
实施例7
本实施例提供了异质性肝祖细胞样细胞库、肝祖细胞样细胞库以及体外三维肝细胞模型。所述肝祖细胞样细胞库为异质性的永生化肝祖细胞样细胞库。
具体的，所述步骤S1中，对得到的所述第一肝祖细胞样细胞系、所述第二肝祖细胞样细胞系、所述第三肝祖细胞样细胞系、所述第四肝祖细胞样细胞系、所述第五肝祖细胞样细胞系以及所述第六肝祖细胞样细胞系分别进行所述冻存处理，得到了所述异质性肝祖细胞样细胞库。
所述肝祖细胞样细胞库由所述第一永生肝祖细胞样细胞株、所述第二永生肝祖细胞样细胞株、所述第三永生肝祖细胞样细胞株、所述第四永生肝祖细胞样细胞株、所述第五永生肝祖细胞样细胞株和所述第六永生肝祖细胞样细胞株组 成。
利用所述肝祖细胞样细胞库建立所述体外三维肝细胞模型。所述体外三维肝细胞模型的构建方法记载在2018年发表在Cell Research(细胞研究)第29卷第1期的“Expansion and differentiation of human hepatocyte-derived liver progenitor-like cells for the study of hepatotropic pathogens”中，在此不做赘述。在所述体外三维肝细胞模型的构建过程中，通过所述第一永生肝祖细胞样细胞株、所述第二永生肝祖细胞样细胞株、所述第三永生肝祖细胞样细胞株、所述第四永生肝祖细胞样细胞株、所述第五永生肝祖细胞样细胞株和所述第六永生肝祖细胞样细胞株分别得到了第一肝细胞模型株、第二肝细胞模型株、第三肝细胞模型株、第四肝细胞模型株、第五肝细胞模型株以及第六肝细胞模型株。
本发明实施例在细胞倒置显微镜下以40倍的放大倍数分别对所述第一人原代肝细胞培养物和经所述转化培养后得到的第一肝祖细胞样细胞系进行了形态观察，得到如图1所示的人原代肝细胞的形态示意图以及图2所示的所述第一肝祖细胞样细胞系的结构示意图。
参照图1和图2，经所述转化培养后，所述第一人原代肝细胞培养物中的人原代肝细胞逐渐转化为具有较规则形态的所述第一肝祖细胞样细胞系，有利于获得稳定的增殖能力。
本发明实施例对所述第一永生肝祖细胞样细胞株、所述第二永生肝祖细胞样细胞株、所述第三永生肝祖细胞样细胞株以及所述第四永生肝祖细胞样细胞株分别进行了40代、30代、20代以及10代的体外增殖培养，以得到如图3所示的增殖性能对比图并统计了相应的倍增时间。
所述体外增殖培养的具体实施方式以及增殖性能和倍增时间的标准方法记载在2018年发表在Cell Research(细胞研究)第29卷第1期的“Expansion and differentiation of human hepatocyte-derived liver progenitor-like cells for the study of hepatotropic pathogens”中，在此不做赘述。
参照图3，在20天的体外增殖培养过程中，所述第一永生肝祖细胞样细胞株、所述第二永生肝祖细胞样细胞株、所述第三永生肝祖细胞样细胞株以及所述第四永生肝祖细胞样细胞株的细胞个数以及增殖速率均相差不大，而所述第一永生肝祖细胞样细胞株、所述第二永生肝祖细胞样细胞株、所述第三永生肝祖细胞样细胞株以及所述第四永生肝祖细胞样细胞株的相应倍增时间分别为29.65±0.4小时、28.32±0.1小时、30.90±0.3小时以及28.92±0.1小时，可见，由实施例1-4的不同供体提供的人原代肝细胞培养物，经过所述转化培养后再进行所述冻存处理后，均能够成功进行永生化建系，且均具有良好的体外增生能力。
本发明实施例在细胞倒置显微镜下以100倍的放大倍数分别对经所述体外增殖培养后的第一永生肝祖细胞样细胞株、所述第二永生肝祖细胞样细胞株、所述第三永生肝祖细胞样细胞株以及所述第四永生肝祖细胞样细胞株进行了形态观察，得到如图4a至图4d所示的对应的形态示意图。
参照图4a至图4d，经所述体外增殖培养后的第一永生肝祖细胞样细胞株、所述第二永生肝祖细胞样细胞株、所述第三永生肝祖细胞样细胞株以及所述第四永生肝祖细胞样细胞株具有相似的形态，其生长特征基本没有受到影响。
本发明实施例在细胞倒置显微镜下以100倍的放大倍数对所述体外三维肝细胞模型中的第一肝细胞模型株进行了形态观察，得到如图5所示的形态示意图。
参照图5，所述第一肝细胞模型株具有较规则的球形结构，表明了良好肝细胞功能。
本发明实施例利用特异质毒性药物厄洛替尼(Erlotinib)对所述体外三维肝细胞模型进行了特异质肝毒性表征。
所述肝毒性表征的具体方法为：
利用二甲基亚砜(Dimethyl sulfoxide，DMSO)配置浓度分别为250μMol/L、62.5μMol/L、15.625μMol/L、3.9μMol/L、0.97μMol/L、0.24μMol/L的Erlotinib溶液，并以DMSO作为空白参比。
使用不同浓度的Erlotinib水溶液对所述体外三维肝细胞模型中的每个肝细胞模型株进行48小时的共培养。
所述共培养结束后，使用生命科技公司(Life Technologies)生产的PrestoBlue TM细胞活性检测试剂分别对经所述共培养后得到的每个培养物的Erlotinib特异质肝毒性进行表征。所述共培养和所述Erlotinib特异质肝毒性表征的具体方法为本领域技术人员的常规手段，在此不做赘述。
图6a至图6f分别为所述第一肝细胞模型株、所述第二肝细胞模型株、所述第三肝细胞模型株、所述第四肝细胞模型株、所述第五肝细胞模型株以及所述第六肝细胞模型株的Erlotinib特异质肝毒性示意图。
参照图6a至图6f，虽然所述第一肝细胞模型株、所述第二肝细胞模型株、所述第五肝细胞模型株和所述第六肝细胞模型株随Erlotinib溶液浓度的增加，细胞活性基本没有显著变化，对Erlotinib没有体现出特异质肝毒性，所述第三肝细胞模型株和所述第四肝细胞模型株随Erlotinib溶液浓度的增加，细胞活性基本有显著的下降趋势，表现除了对Erlotinib的特异质肝毒性，继而有利于开展对Erlotinib特异性毒性的研究。
实施例8
本发明实施例8还提供了所述永生肝祖细胞样细胞株在生物人工肝方面的应用。
具体的，采用保藏编号为CCTCC NO：C2019125的永生肝祖细胞样细胞株进行所述应用。所述应用包括：细胞增殖、动物模型的构建、体外循环系统的建立以及取样检测。
所述细胞增殖包括：将保藏编号为CCTCC NO：C2019125的永生肝祖细胞样细胞株负载于载体，然后通过液气交互式生物反应器进行细胞培养，以实现肝细胞增殖。
图7为本发明实施例8的液气交互式生物反应器的结构示意图。
参照图7，液气交互式生物反应器7具有内部相通的容器本体71和波纹管73，以及设置于所述容器本体71的出液管72和进液管74。将保藏编号为CCTCC NO：C2019125的永生肝祖细胞样细胞株负载于空白载片形成负载载片，然后放置于所述容器本体71，将2019年细胞研究(Cell Research)的第29卷第1期第8页至第22页公开的内容中使用的细胞培养液通过所述进液管74引入所述波纹管73，依靠驱动所述波纹管73进行升降运动带动所述细胞培养液周期性地浸没所述负载载片以进行液相物质交换，且所述负载载片能够与所述容器本体71内空气中的氧气进行气相物质交换，以进行所述细胞增殖。所述空白载片的质量为11克。
具体的，控制所述波纹管73的进行升降运动的速率为0.5-5毫米/秒，在所述波纹管73的每个升降周期中，控制所述负载载片进行液相物质交换的时间为100-300秒，进行气相物质交换的时间为5-120秒。所述液相物质交换的时间指所述负载载片浸没于所述细胞培养液的时间，所述气相物质交换的时间指所述负载载片暴露于空气中的时间。所述细胞增殖的时间为14天。
在进行所述细胞增殖的过程中，每24小时更换一次细胞培养液，以有利于细胞有效进行三维扩增。
经14天的所述细胞增殖后，负载于所述载片的肝细胞数目从培养前的0.5×10 9个扩增为2.5×10 9个，对经所述细胞增殖后得到的增殖细胞在细胞倒置显微镜下以100倍的放大倍数进行观察，得到图8所示的形态示意图。参照图8，可见得到的增殖细胞具有较规则的形态结构，有利于发挥良好的细胞功能。
本发明实施例8进一步考察了经不同增殖时间得到的增殖细胞的不同功能基因的基因表达水平，以考察细胞功能。
具体的，采用上海飞捷生物技术有限公司生产的货号为220010的RNAfast200试剂盒抽提所述增殖细胞的RNA，然后采用赛默飞世尔科技(Invitrogen)公司生产的货号为18064014的反转录酶将得到的RNA分别反转录为cDNA，最后通 过荧光定量PCR分别表达相关功能基因，所述荧光定量PCR的具体表达方法为本领域技术人员的常规手段，在此不做赘述。
相关的功能基因分别为氨甲酰磷酸合成酶1(carbamoyl phosphate synthetase 1,CPS1)、α1-抗胰蛋白酶(Alpha-1-antitrypsin，AAT)、白蛋白(Albumin，Alb)、药物代谢酶CYP3A4、蛋白质编码基因GSTA2以及多药耐药相关蛋白MRP4。经2天的细胞增殖后得到的增殖细胞与经14天的细胞增殖后得到的增殖细胞在CPS1、AAT、Alb、CYP3A4、GSTA2以及MRP4方面的平均基因表达水平结果请参见表1。
表1
功能基因 CPS1 AAT Alb CYP3A4 GSTA2 MRP4
第2天 1 1 1 1 1 1
第14天 5 2 6 4 10 1.5
从表1中可以看到，经14天的细胞增殖后，CPS1、AAT、Alb和GSTA2的平均基因表达水平均有显著提升，增殖细胞表现出了良好的蛋白合成功能；GSTA2以及MRP4方面的平均基因表达水平的显著上升则说明增殖细胞具有良好的药物代谢能力。
所述动物模型的构建包括：将D-氨基半乳糖(D-(+)-Galactosamine hydrochloride，D-gal)溶于质量浓度为5％的葡萄糖水溶液，定容至0.5g/mL后用1mol/L的氢氧化钠水溶液调节pH值至6.8，形成D-gal溶液；使用设置有平均孔径为0.22微米滤膜的针筒式过滤器所述D-gal溶液进行过滤除菌。
六月龄，体重为20-30公斤的健康实验小猪在全身麻醉前的8小时禁食，在全身麻醉前的4小时禁止饮水；全身麻醉后在超声引导下采用塞尔丁格(Sedinger)穿刺技术对小猪的股静脉和颈外静脉进行穿刺置管；待小猪恢复自主呼吸后，按照每公斤小猪注射0.5g D-gal的剂量向恢复自主呼吸的小猪体内推注所述 D-gal溶液，并于30分钟内完成所述推注，以建立急性肝衰竭的生物模型。
取全血进行检测，当检测结果显示实验小猪的丙氨酸转氨酶(Alanine aminotransferase，ALT)和天冬氨酸转氨酶(Aspartate aminotransferase，AST)分别高于200U/L和1000U/L，国际标准化比值(international normalized ratio，INR)达到2-3，血氨值高于45μmol/L，则判断急性肝衰竭的生物模型建立成功。
图9为本发明实施例8的体外循环系统的结构示意图。
参照图9，所述体外循环系统的建立包括：
所述急性肝衰竭的生物模型建立成功后，将实验小猪的血液通过股静脉穿刺导管接入第一导管91，以通过泵送的方式输送至血浆分离器92，同时通过所述第一导管91向所述血浆分离器92中加注肝素，全身肝素剂量控制为每公斤小猪100单位。
所述血浆分离器92将分离出的血浆通过第二导管93泵送入所述液气交互式生物反应器7内，所述液气交互式生物反应器7内放置有经所述细胞增殖后得到的载片，然后通过所述液气交互式生物反应器7控制血浆和载片之间的液相物质交换和气相物质交换，具体的，参照图7，控制所述波纹管73的进行升降运动的速率为2毫米/秒，在所述波纹管73的每个升降周期中，控制所述负载载片进行液相物质交换的时间为120秒，进行气相物质交换的时间为10秒。具体的控制方法请参见所述细胞增殖的叙述，在此不做赘述。
将得到的解毒后的血浆经第三导管94泵送入所述血浆分离器92以与血细胞重新混合形成净化血液，通过第四导管95回流至实验小猪体内，以完成循环治疗过程。所述循环治疗的总时间为3h。
继续饲养经所述循环治疗后的实验小猪，所述循环治疗结束后的65小时实验小猪即可稳定站立并逐渐恢复饮食，96小时后仍能够存活。
所述取样检测包括：在所述循环治疗结束后的72小时内的不同时间对实验小猪进行抽血检测，考察血清生化指标和肝脏凝血能力指标。
另外，本发明实施例提供了对比例1，将实验小猪的血液经型号为BS330的胆红素吸附柱以及中空纤维管过滤后，通过图7所示的体外循环系统流经空白的所述液气交互式生物反应器7，以进行3小时的循环治疗。空白的所述液气交互式生物反应器7中的载片为空白载片，其余具体的控制过程请参见前述。
其中，实施例8和对比例1的血清生化指标为ALT含量、AST含量、血清氨(Ammonia)含量、总胆红素(Total bilirubin，TBiL)含量、乳酸脱氢酶(Lactate dehydrogenase，LDH)含量和Alb含量含量，具体数值请参见表2。
表2
Figure PCTCN2020086659-appb-000001
表2中ALT和AST的单位为U/L，Ammonia、TBiL和LDH的单位为μmol/L，Alb的单位为g/L。
参见表2，所述循环治疗结束后的72小时，对比例1和实施例8的ALT升高的同时AST也同时升高，但实施例8的ALT和AST升高的程度低于对比例1的对应血清生化指标的升高程度。对比例1和实施例8的血清氨含量均升高，但实施例8的血清氨含量升高的程度也低于对比例的对应血清生化指标的升高程度。
进一步的，从所述循环治疗结束后的72小时内的不同时间测得的数据来看，对比例1的ALT在所述循环治疗结束后的24小时后由200U/L急剧上升至2500U/L，然后又急剧下降至700U/L，而实施例8的ALT始终维持在200U/L以内，且在所述循环治疗结束后的48至72小时内有缓慢下降的趋势。
而对比例1和实施例8的Ammonia在所述循环治疗结束后的24小时内分别增加至80μmol/L和50μmol/L，然后，实施例8的Ammonia持续降低直至不高于10μmol/L，而对比例1的Ammonia在经过24小时的缓慢降低后又显著增加至450μmol/L。
综合ALT、AST和Ammonia的数据情况可知，经实施例8的由永生肝祖细胞样细胞株增殖形成的增殖细胞具有一定的解毒能力，相比于对比例1而言能够有效降低肝损伤的程度。
参照表2，所述循环治疗结束后的72小时，对于实施例8而言，TBiL有所升高，但升高的幅度不大，而对比例1的TBiL却出现了显著的升高。
进一步的，从所述循环治疗结束后的72小时内的不同时间测得的数据来看，对比例1的TBiL在所述循环治疗结束后的48小时内显著上升至60μmol/L，然后又下降至56.7μmol/L。实施例8的TBiL始终不超过3.5μmol/L。
从TBiL的数据情况可知，经实施例8的由永生肝祖细胞样细胞株增殖形成的增殖细胞解毒后，实施例8的实验小猪表现出的肝脏分泌和排泄功能要优于对比例1的实验小猪。
参照表2，所述循环治疗结束后的72小时，对于实施例8而言，Alb下降的幅度很低，从所述循环治疗结束后的72小时内的不同时间测得的数据来看，实施例8的Alb在36-40g/L之间变动。
参照表2，所述循环治疗结束后的72小时，对比例1的Alb下降的幅度要高于实施例8，从所述循环治疗结束后的72小时内的不同时间测得的数据来看，对比例1的Alb在29-35g/L之间变动。
从Alb的数据情况可以看到，经实施例8的由永生肝祖细胞样细胞株增殖形成的增殖细胞解毒后，实施例8的实验小猪的肝脏合成和储备蛋白质的能力要优于对比例的实验小猪。
参照表2，所述循环治疗结束后的72小时，对比例1的LDH远高于实施例8，显示出了异常的活性，从所述循环治疗结束后的72小时内的不同时间测得的数据来看，对比例1的LDH从所述循环治疗结束后就保持着显著的上升趋势，而实施例8的LDH虽然也有上升的趋势，但增长幅度远低于对比例。
从LDH的数据情况可以看到，对比例1的实验小猪的肝脏相比实施例8的实验小猪的肝脏具有更严重的肝损伤。
所述肝脏凝血能力指标包括凝血酶原时间(Prothrombin time，PT)、凝血酶时间(Thrombin Time，TT)、活化部分凝血活酶时间(Activated Partial Thromboplastin Time，APTT)和INR。具体的测试手段为本领域技术人员的常规技术手段，在此不做赘述。
具体的，对比例1的PT自所述循环治疗结束后的第24小时至第72小时，从20秒显著上升至150秒，而实施例8的PT始终不超过30秒，且自循环治疗结束后的第24小时至第72小时，PT有缓慢下降的趋势，并最终下降至12秒。
对比例1的INR自所述循环治疗结束后的第24小时至第72小时，从1.78显著上升至8，而实施例8的INR始终不超过2.6，且自循环治疗结束后的第24小时至第72小时，APTT有缓慢下降的趋势，并最终下降至1.05。
对比例1的实验小猪和实施例8的实验小猪在循环治疗结束后的48小时内测得的TT相差不大，且均呈现出增加的趋势，具体为从20秒左右上升至40秒左右，在循环治疗结束后的72小时，实施例8的TT下降至21秒，而对比例1的TT继续上升至50秒左右。
对比例1的实验小猪和实施例8的实验小猪测得的APTT在循环治疗结束后的24小时内均呈现缓慢增加的趋势，具体的，对比例1的APTT增加至20秒， 实施例8的APTT增加至28秒；在循环治疗结束后的24小时至72小时内，对比例1的APTT显著增加至60秒，而实施例8的APTT具有下降趋势，且最终下降至18秒。
综合上述肝脏凝血能力指标可见，对比例1的实验小猪的肝脏合成凝血因子的能力显著降低，肝损伤的程度高。
综上所述，使用实施例8的由永生肝祖细胞样细胞株增殖形成的增殖细胞对血浆进行解毒，对前述的血清生化指标和肝脏凝血能力指标趋向正常有积极的辅助作用。
另外，更重要的是，向所述液气交互式生物反应器7灌注实验小猪的血浆后，实验小猪的血浆与经永生肝祖细胞样细胞株增殖形成的增殖细胞经过前述的气相物质交换和液相物质交换后，从得到的净化血浆中检测到了至少一种外源性人类生长因子，由此证明本发明实施例的所述永生肝祖细胞样细胞株增殖形成的细胞应用于生物人工肝治疗的过程中能够分泌至少一种外源性人类生长因子。
具体的，所述至少一种外源性人类生长因子包括人源的肝细胞生长因子(Human hepatocyte growth factor，HumanHGF)、人源的转化生长因子-α(Human Transforming Growth Factor-α，Human TGF-α)和人源的白介素-6(Human Interleukin-6，Human IL-6)中的任意一种或多种。
具体的，所述循环治疗进行的3h内，实施例8和对比例1在不同的时间从所述液气交互式生物反应器7的血浆中采样分析得到的Human HGF、Human TGF-α、Human IL-6、尿素以及乳酸含量的数值请参见表3。
表3
Figure PCTCN2020086659-appb-000002
Figure PCTCN2020086659-appb-000003
参照表3，本发明实施例8中，所述至少一种外源性人类生长因子为Human HGF、Human TGF-α和Human IL-6，有助于对早期肝脏的恢复起到积极的促进作用。
另外，检测到所述净化血浆中的尿素水平升高，说明本发明实施例的所述永生肝祖细胞样细胞株增殖形成的细胞有助于解毒实验小猪血浆中的氨类毒性物质，而所述净化血浆中的乳酸浓度降低，进一步证实了本发明实施例的所述永生肝祖细胞样细胞株增殖形成的细胞具有肝代谢功能。
实施例9
本发明实施例9提供了所述永生化肝祖细胞样细胞株在肝细胞移植方面的应用，包括小鼠进行肝衰竭模型的构建、体内生物反应器的制备和移植。
所述小鼠急性肝衰竭模型的构建包括：提供包含四氯化碳的橄榄油作为诱导剂，以200微升10％四氯化碳对NSG重度免疫缺陷实验小鼠进行皮下注射。次日，眼眶静脉取血样作为对照体重减轻1-3g为宜，状态较前日略有变差，以完成所述小鼠模型的构建。
所述体内生物反应器的制备包括：将海藻酸钠水溶液和实施例8中由保藏编号为CCTCC NO：C2019125的永生化肝祖细胞样细胞株制备的增殖细胞所形成的悬浮细胞团制备细胞球微囊，并作为所述体内生物反应器使用。具体的制备方法包括：提供以无菌生理盐水配置的质量体积浓度为1.5％的海藻酸钠水溶液；将所述海藻酸钠水溶液与所述悬浮细胞团均匀混合，使形成的每毫升混合液中含有3×10 6个细胞；使用配置有D250微米无菌喷嘴的型号为IE-50R的封装机将所述混合液喷入质量浓度为1.2％的氯化钙水溶液中交联10分钟，得到湿态细胞球微囊；使用生理盐水对所述湿态细胞球微囊进行洗涤以去除游离态钙离子后再使用去离子水进行洗涤，以得到所述细胞球微囊。
本发明一些实施例中，经去离子水洗涤后的细胞球微囊的平均直径不超过500微米。
图10为本发明一些实施例的细胞球微囊的电镜照片。参照图10，由于海藻酸钠具有良好的生物相容性和pH敏感性，可在温和的条件下快速形成水凝胶，并通过盐键、氢键或疏水作用结合生物活性分子，细胞球微囊101的内部包覆有增殖细胞团102，形成了所述体内生物反应器。所述体内生物反应器的直径约为85微米。
所述体内生物反应器的移植包括：将所述体内生物反应器使用生理盐水进行重新分散，然后对所述鼠模型进行腹腔移植。
本发明实施例还提供了对比例2，使用分散后的不含增殖细胞的微囊对所述鼠模型进行腹腔移植。所述不含增殖细胞的微囊制备方法与所述细胞球微囊制备方法的区别在于：所述海藻酸钠水溶液中不添加所述悬浮细胞团。
所述腹腔移植结束后的7天，将实施例9的存活的实验小鼠安乐死后进行解剖观察，发现所述体内生物反应器分散在整个腹腔，并松散连接到肠系膜和网膜。
经所述内腹移植后，使用卡普兰-迈尔(Kaplan-meier)法对实施例9和对比例 2的实验小鼠进行生存分析，具体的分析方法为本领域技术人员的常规技术手段，在此不做赘述。
所述生存分析的结果表明：在所述内腹移植结束后第2天，实施例9的生存率下降至70％；随着观察时间延长至所述内腹移植结束后的第7天，实施例9的生存率维持70％不变，健康状况评定为半依赖级；对比例2的生存率每天下降15-20％，在所述内腹移植结束后第7天，对比例2的生存率下降至15％，健康状况评定为病重。
所述腹腔移植结束后以及结束后的第1、3和7天分别对实施例9和对比例2的实验小鼠检测血清ALT和AST，结果表明，所述腹腔移植结束后的第1天，实施例9的血清ALT和AST均下降70-80％，而对比例2的血清ALT和AST均下降30-40％，所述腹腔移植结束后的第3-7天，实施例9和对比例2的存活小鼠血清ALT和AST均下降至正常水平。
综合上述分析可见，所述永生肝祖细胞样细胞株制备的增殖细胞有助于缓解所述鼠模型的肝脏损伤程度。
虽然在上文中详细说明了本发明的实施方式，但是对于本领域的技术人员来说显而易见的是，能够对这些实施方式进行各种修改和变化。但是，应理解，这种修改和变化都属于权利要求书中所述的本发明的范围和精神之内。而且，在此说明的本发明可有其它的实施方式，并且可通过多种方式实施或实现。

Claims (13)

  1. 一种肝祖细胞样细胞库的构建方法，其特征在于，包括：
    S1：提供不同供体来源的人原代肝细胞培养物，对每个供体来源的人原代肝细胞培养物在0.5×10 4-5×10 4个/平方厘米的接种密度下进行7-14天的转化培养，然后对经所述转化培养后得到的每个供体来源的肝祖细胞样细胞系进行冻存处理，以得到异质性肝祖细胞样细胞库；
    S2：对每个供体来源的肝祖细胞样细胞系解冻后进行增殖培养，以分别得到不同供体来源的贴壁细胞，每个供体来源的贴壁细胞的汇合率为70％-90％；
    S3：对每个供体来源的贴壁细胞依次进行第一传代处理和病毒感染，并在所述病毒感染的过程中进行培养基置换；
    S4：对经所述病毒感染后得到的每个供体来源的培养物进行传代次数为2或3的第二传代处理，通过筛选培养基对经所述第二传代处理后得到的培养物进行连续筛选培养，以得到不同供体来源的感染培养物；
    S5：对每个供体来源的感染培养物进行传代比例为1:2-1:4，传代次数为5-10的连续传代培养，以得到所述肝祖细胞样细胞库。
  2. 根据权利要求1所述的构建方法，其特征在于，所述步骤S1中，所述转化培养结束后，对经所述转化培养后得到的培养物依次进行2或3次的扩增培养后，再执行所述冻存处理。
  3. 根据权利要求1所述的构建方法，其特征在于，所述步骤S2中，所述肝祖细胞样细胞系在0.5×10 4-5×10 4个/平方厘米的接种密度下进行所述增殖培养，所述增殖培养开始后的三天内，每天进行培养基置换。
  4. 根据权利要求1所述的构建方法，其特征在于，所述步骤S3中，所述每个供体来源的贴壁细胞在2×10 4-4×10 4个/平方厘米的接种密度下进行24小时的所述第一传代处理。
  5. 根据权利要求1所述的构建方法，其特征在于，所述步骤S3中，经所述第一传代处理后得到的培养物在2×10 4-4×10 4个/平方厘米的接种密度下进行所述病毒感染。
  6. 根据权利要求5所述的构建方法，其特征在于，向经所述第一传代处理后得到的培养物中加入DMEM/12培养基、慢病毒和聚凝胺悬液后的6-12小时后进行培养基置换，然后继续培养24-72小时，以完成所述病毒感染，所述慢病毒与所述贴壁细胞的数量比为0.5-60。
  7. 根据权利要求1所述的构建方法，其特征在于，所述步骤S4中，经所述病毒感染得到的每个供体来源的培养物在2×10 4-4×10 4个/平方厘米的接种密度下进行5-7天的所述第二传代处理，所述第二传代处理的过程中，每2-3天进行一次培养基置换。
  8. 根据权利要求1所述的构建方法，其特征在于，所述筛选培养基为TEM培养基。
  9. 根据权利要求1所述的构建方法，其特征在于，所述步骤S5中，所述每个供体来源的感染培养物以2×10 4-4×10 4个/平方厘米的接种密度进行所述连续传代培养。
  10. 一种肝祖细胞样细胞库的应用，其特征在于，通过如权利要求1-9中任一项所述的构建方法得到的异质性永生肝祖细胞样细胞库构建体外三维肝细胞模型，然后利用所述体外三维肝细胞模型检测药物的特异质肝毒性。
  11. 一种如权利要求1-9中任意一项所述的构建方法制备的永生肝祖细胞样细胞株，其特征在于，所述永生肝祖细胞株的分类命名为81.5，保藏于中国典型培养位保藏中心，保藏编号为CCTCC NO：C2019125。
  12. 一种如权利要求1-9中的任意一项所述的构建方法制备的永生肝祖细胞样细胞株在生物人工肝和肝细胞移植方面的应用，其特征在于，所述永生肝祖细胞样细胞株增殖形成的细胞应用于生物人工肝治疗的过程中分泌至少一种外源性 人类生长因子。
  13. 根据权利要求12所述的应用，其特征在于，所述至少一种外源性人类生长因子包括人源的肝细胞生长因子、人源的转化生长因子-α和人源的白介素-6中的任意一种或多种。
PCT/CN2020/086659 2019-07-11 2020-04-24 肝祖细胞样细胞库的构建方法及其制备的细胞株与应用 WO2021004129A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP20830058.2A EP3978600A4 (en) 2019-07-11 2020-04-24 METHOD FOR GENERATING A LIVER PROGENITOR LIKE CELL BANK, CELL LINES PREPARED THEREFROM AND THEIR USE
US17/622,838 US20220411758A1 (en) 2019-07-11 2020-04-24 Method for constructing hepatic progenitor cell-like cell bank, cell lines prepared therefrom and application thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910623436.9 2019-07-11
CN201910623436 2019-07-11

Publications (1)

Publication Number Publication Date
WO2021004129A1 true WO2021004129A1 (zh) 2021-01-14

Family

ID=71433792

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/086659 WO2021004129A1 (zh) 2019-07-11 2020-04-24 肝祖细胞样细胞库的构建方法及其制备的细胞株与应用

Country Status (4)

Country Link
US (1) US20220411758A1 (zh)
EP (1) EP3978600A4 (zh)
CN (1) CN111394391B (zh)
WO (1) WO2021004129A1 (zh)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008002523A2 (en) * 2006-06-28 2008-01-03 Vesta Therapeutics, Inc. Matrix and method for isolation of hepatic progenitor cells
CN101228266A (zh) * 2005-05-26 2008-07-23 弗雷森纽斯医疗护理德国有限责任公司 肝祖细胞
CN101275121A (zh) * 2007-03-26 2008-10-01 芦银雪 体外培养扩增的人肝脏祖先细胞及其制备方法
CN102119031A (zh) * 2008-06-11 2011-07-06 弗雷森纽斯医疗护理德国有限责任公司 肝祖细胞的条件培养基
CN102234626A (zh) * 2010-05-06 2011-11-09 国玺干细胞应用技术股份有限公司 肝祖细胞及其应用
CN108300688A (zh) 2018-02-07 2018-07-20 上海赛立维生物科技有限公司 原代肝细胞分离和培养方法
CN108330099A (zh) 2017-03-22 2018-07-27 上海赛立维生物科技有限公司 个性化肝细胞的培养和扩增方法及其应用

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NZ513150A (en) * 1999-01-19 2004-09-24 Univ North Carolina Human liver progenitors
US7456017B2 (en) * 1999-10-01 2008-11-25 University Of North Carolina At Chapel Hill Processes for clonal growth of hepatic progenitor cells
US20030096411A1 (en) * 1999-12-07 2003-05-22 George Michalopoulos Novel long-term three-dimensional tissue culture system
RU2346981C2 (ru) * 2002-07-19 2009-02-20 Веста Терапьютикс, Инк. Способ получения жизнеспособных клеток печени человека, в том числе печеночных стволовых клеток/клеток-предшественников
US8017391B2 (en) * 2006-03-16 2011-09-13 Univesity of Florida Research Foundation, Inc. Human liver cancer cell line
WO2011009294A1 (zh) * 2009-07-23 2011-01-27 北京华源博创科技有限公司 通过诱导分化获得肝脏细胞、肝脏内胚层细胞和肝脏前体细胞的方法
MX369514B (es) * 2013-08-28 2019-11-11 Promethera Biosciences S A /N V Metodo para producir celulas progenitoras hepaticas adultas.
CN109337858B (zh) * 2018-09-20 2022-03-15 中国人民解放军第二军医大学 用于乙肝病毒感染的原代肝细胞来源的肝前体样细胞模型、制备方法及应用
CN110438157B (zh) * 2019-08-05 2020-11-24 上海赛立维生物科技有限公司 肝前体样细胞系、构建方法以及在生物人工肝领域的应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101228266A (zh) * 2005-05-26 2008-07-23 弗雷森纽斯医疗护理德国有限责任公司 肝祖细胞
WO2008002523A2 (en) * 2006-06-28 2008-01-03 Vesta Therapeutics, Inc. Matrix and method for isolation of hepatic progenitor cells
CN101275121A (zh) * 2007-03-26 2008-10-01 芦银雪 体外培养扩增的人肝脏祖先细胞及其制备方法
CN102119031A (zh) * 2008-06-11 2011-07-06 弗雷森纽斯医疗护理德国有限责任公司 肝祖细胞的条件培养基
CN102234626A (zh) * 2010-05-06 2011-11-09 国玺干细胞应用技术股份有限公司 肝祖细胞及其应用
CN108330099A (zh) 2017-03-22 2018-07-27 上海赛立维生物科技有限公司 个性化肝细胞的培养和扩增方法及其应用
CN108300688A (zh) 2018-02-07 2018-07-20 上海赛立维生物科技有限公司 原代肝细胞分离和培养方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
"Expansion and differentiation of human hepatocyte-derived liver progenitor-like cells for the study of hepatotropic pathogens", CELL RESEARCH, vol. 29
"Expansion and differentiation of human hepatocyte-derived liver progenitor-like cells for the study of hepatotropic pathogens", CELL RESEARCH, vol. 29, 2018
HANG HUALIAN; ZHANG LEI; SHI XIAOLEI: "Establishment and biological study of immortalized human hepatocyte cell line", JIANGSU YIYAO = JIANGSU MEDICAL JOURNAL, vol. 37, no. 22, 30 November 2011 (2011-11-30), pages 2624 - 2627, XP055778351, ISSN: 0253-3685, DOI: 10.19460/j.cnki.0253-3685.2011.22.006 *
See also references of EP3978600A4
WEBER, A.: "Immortalization of hepatic progenitor cells", PATHOLOGIE BIOLOGIE, vol. 52, 31 December 2004 (2004-12-31), XP055103995, DOI: 20200710203708 *

Also Published As

Publication number Publication date
EP3978600A1 (en) 2022-04-06
US20220411758A1 (en) 2022-12-29
CN111394391A (zh) 2020-07-10
CN111394391B (zh) 2022-12-06
EP3978600A4 (en) 2022-08-03

Similar Documents

Publication Publication Date Title
JP3692147B2 (ja) フィルターデバイス
US6875605B1 (en) Modular cell culture bioreactor and associated methods
CN105985985B (zh) Crispr技术编辑并用igf优化的异体间充质干细胞的制备方法及在治疗心梗中应用
CN100441682C (zh) 获得活的人肝细胞,包括肝干/祖细胞的方法
US9650609B2 (en) Bioartificial liver system
EP3245284A1 (en) Pluripotent stem cell expansion and passage using a rocking platform bioreactor
CN107164326B (zh) 一种3D培养自体脂肪MSCs来源的神经前体细胞的方法
KR101293364B1 (ko) 조직공학적 인간 각막 내피의 재건 방법
WO2010105204A2 (en) Bioartificial liver
TW200916583A (en) Cell expansion
US8735143B2 (en) Hepatic lobule-like bioreactor
CN105769910B (zh) 一种人羊膜间充质干细胞的应用
Teng et al. Treatment of acute hepatic failure in mice by transplantation of mixed microencapsulation of rat hepatocytes and transgenic human fetal liver stromal cells
TW201437365A (zh) 體外血腦屏障模型之建立方法
CN104818242B (zh) 原代肝细胞的分离制备方法
Shito et al. Efficacy of an extracorporeal flat-plate bioartificial liver in treating fulminant hepatic failure
US9017657B2 (en) Islet cell cluster produced from human umbilical cord mesenchymal stem cells
WO2021004129A1 (zh) 肝祖细胞样细胞库的构建方法及其制备的细胞株与应用
CN104845930B (zh) 用于分离原代肝细胞的联合用试剂
CN112868641A (zh) 一种胰岛细胞的冻存方法、复苏方法及重构方法
CN109576308A (zh) 一种提高人类干细胞来源肝样细胞解毒功能的方法及其应用
CN106267425A (zh) Aids免疫吸附治疗仪
CN111849904B (zh) 神经母细胞瘤类器官的培养基、培养方法及移植体
CN107519207A (zh) 一种免疫抑制细胞制剂及其制备方法和应用
CN110607279B (zh) 一种原代肿瘤细胞的3d培养体系及其培养方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20830058

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2020830058

Country of ref document: EP

Effective date: 20211229