WO2022181660A1 - スクリーニング方法に使用するためのヒト脂肪肝モデル細胞 - Google Patents

スクリーニング方法に使用するためのヒト脂肪肝モデル細胞 Download PDF

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WO2022181660A1
WO2022181660A1 PCT/JP2022/007494 JP2022007494W WO2022181660A1 WO 2022181660 A1 WO2022181660 A1 WO 2022181660A1 JP 2022007494 W JP2022007494 W JP 2022007494W WO 2022181660 A1 WO2022181660 A1 WO 2022181660A1
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cells
vldl
medium
fatty liver
human
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French (fr)
Japanese (ja)
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真生 高橋
雅和 加国
奈美 吉川
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Phoenixbio Co Ltd
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Phoenixbio Co Ltd
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Priority to CN202280017368.1A priority Critical patent/CN116917462A/zh
Priority to EP22759695.4A priority patent/EP4299720A4/en
Priority to US18/278,605 priority patent/US20250263667A1/en
Priority to CA3211975A priority patent/CA3211975A1/en
Priority to JP2023502465A priority patent/JP7449626B2/ja
Publication of WO2022181660A1 publication Critical patent/WO2022181660A1/ja
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    • 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
    • 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

Definitions

  • the present invention provides a human fatty liver model for use in screening methods for substances having effects on dyslipidemia including fatty liver, using the increase or decrease in very low density lipoprotein (VLDL) in the culture supernatant as an index. Regarding cells.
  • VLDL very low density lipoprotein
  • Fatty liver is a general term for diseases in which lipids such as neutral fat accumulate excessively in hepatocytes, causing liver damage.
  • lipids such as neutral fat accumulate excessively in hepatocytes, causing liver damage.
  • accumulation of lipid droplets is observed in 1/3 or more of the hepatocytes that constitute the hepatic lobules.
  • the frequency of fatty liver tends to increase year by year due to changes in eating habits and lifestyle habits.
  • NASH non-alcoholic fatty liver disease
  • NASH non-alcoholic steatohepatitis
  • liver cancer liver cancer
  • a non-human animal model showing the symptoms of fatty liver has been created to study the onset mechanism of fatty liver and its prevention and treatment.
  • a non-human animal model showing symptoms of fatty liver is prepared by transplanting human hepatocytes into a liver-damaged, immunodeficient non-human animal.
  • symptoms of fatty liver such as large lipid droplets and liver steatosis are observed.
  • an in vitro evaluation system for human fatty liver that is, a human fatty liver model cell, is desired in order to efficiently study the onset mechanism of fatty liver and its prevention and treatment.
  • human hepatocytes derived from fatty liver in vitro, they found that the cells caused disappearance of lipid droplets and the like, and that the symptoms of fatty liver could not be maintained. More specifically, human hepatocytes in which symptoms of fatty liver such as accumulation of lipid droplets are observed are separated and recovered from the fatty liver excised from the non-human animal model exhibiting symptoms of fatty liver described above. was cultured in vitro, the cells caused loss of lipid droplets, etc., and found that the symptoms of fatty liver could not be maintained.
  • the present invention provides a screening method for a substance that can maintain the symptoms of fatty liver such as the accumulation of lipid droplets in human hepatocytes derived from fatty liver and has an effect on dyslipidemia such as fatty liver.
  • An object of the present invention is to provide new human fatty liver model cells that can be used for
  • the present inventors have made intensive studies and found that culturing fatty liver-derived human hepatocytes in a medium containing dimethylsulfoxide results in accumulation of lipid droplets, secretion and accumulation of lipids, The inventors have found that the expression of fatty liver-related genes and the like can be observed, and that human fatty liver model cells that maintain the symptoms of fatty liver can be obtained.
  • the culture supernatant of the human fatty liver model cell contains very low density lipoprotein (VLDL) and low density lipoprotein (LDL), and the VLDL is contained more than the LDL,
  • VLDL very low density lipoprotein
  • LDL low density lipoprotein
  • the present inventors have found that it is possible to screen for substances that are effective against dyslipidemia such as fatty liver by using the increase or decrease in the VLDL content in the culture supernatant as an index.
  • a human fatty liver model cell for use in a method of screening for substances having effects on dyslipidemia including fatty liver, using the increase or decrease in very low density lipoprotein (VLDL) in the culture supernatant as an index. and A cell, characterized in that the culture supernatant of said cell contains VLDL and low density lipoprotein (LDL), said VLDL being higher than said LDL.
  • VLDL very low density lipoprotein
  • LDL low density lipoprotein
  • [3] The cell of [1] or [2], wherein the VLDL comprises Large-VLDL, Medium-VLDL, and Small-VLDL, and the Large-VLDL accounts for 70% by mass or more of the total VLDL.
  • [4-2] The amount of lipoprotein (more preferably triglyceride) contained in the culture supernatant is in the culture supernatant of fatty liver-derived human hepatocytes cultured under the same conditions except that it does not contain dimethyl sulfoxide
  • the cell according to any one of [1] to [4], which is 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, or 9-fold or more the amount of lipoproteins (more preferably triglycerides) contained therein.
  • [5] The cells of [4], [4-1] or [4-2], wherein the fatty liver-derived human hepatocytes are collected from a chimeric non-human animal having human hepatocytes.
  • a screening method for a substance having an effect on dyslipidemia comprising: A step of administering a test substance to the cells of any one of [1] to [5], and A method comprising comparing the amount of very low density lipoprotein (VLDL) in the culture supernatant between cells treated with a test substance and cells not treated.
  • VLDL very low density lipoprotein
  • human fatty liver model cells in which accumulation of lipid droplets, secretion/accumulation of lipids, expression of fatty liver-related genes, etc. are observed, and very low density lipoprotein (VLDL) in the culture supernatant Human fatty liver model cells can be provided for use in a method of screening for substances having effects on dyslipidemia including fatty liver, using the increase or decrease in the index.
  • VLDL very low density lipoprotein
  • FIG. 1 shows photographs of monolayer cell cultures of PXB-cells cultured for 5 days in medium B (DMSO(+)) or medium C (DMSO(-)).
  • PXB-cells left) cultured in medium B (DMSO(+)); PXB-cells (right) cultured in medium C (DMSO(-)).
  • Figure 2 shows the measurement results of the total neutral fat (triglyceride) content of lipoproteins in the culture supernatant of PXB-cells cultured for 5 days in medium B (DMSO (+)) or medium C (DMSO (-)). show. The results are shown as relative values with the content in the culture supernatant of PXB-cells cultured in medium B (DMSO(+)) being "100".
  • FIG. 1 shows photographs of monolayer cell cultures of PXB-cells cultured for 5 days in medium B (DMSO(+)) or medium C (DMSO(-)).
  • PXB-cells left) cultured in medium B (DMSO(
  • FIG. 3 shows PXB-cells cultured for 5 days, 9 days, 12 days, and 14 days in medium B (DMSO (+)), HepG2 cells, and total cholesterol of lipoproteins in the culture supernatant of HuH7 cells (left ) and total triglyceride (right) content.
  • Figure 4 shows PXB-cells cultured for 5 days, 9 days, 12 days, and 14 days in medium B (DMSO (+)), HepG2 cells, and lipoproteins in the culture supernatant of HuH7 cells (4 types of sub group) cholesterol (left) and neutral fat (triglyceride) (right) content analysis results.
  • FIG. 5 shows intracellular total cholesterol (left) and total cholesterol in PXB-cells, HepG2 cells, and HuH7 cells cultured in medium B (DMSO (+)) for 5 days, 9 days, 12 days, and 14 days. The results of measurement of sex fat (triglyceride) (right) content are shown.
  • FIG. 6 shows the measurement results of expression levels of fatty liver-related genes (FASN, SREBF1, G6PC) in PXB-cells cultured in medium B (DMSO(+)) for 3 days and 6 days. The results are shown as relative values with the expression level of each gene in PXB-cells cultured for 3 days in medium B (DMSO(+)) being "1".
  • FIG. 7 shows the results of analyzing the neutral fat (triglyceride) content in each subgroup of lipoproteins in the culture supernatant of PXB-cells cultured for 2 days in medium D containing 0.75 mM oleic acid (A), and VLDL of the culture supernatant was analyzed for the neutral fat (triglyceride) content of each size of Large, Medium, and Small (B).
  • A 0.75 mM oleic acid
  • B VLDL of the culture supernatant
  • Figure 9 shows the result of analysis of the neutral fat (triglyceride) content in each lipoprotein subgroup in the culture supernatant of PXB-cells cultured for 2 days in medium D containing 10 ⁇ M, 50 ⁇ M, and 100 ⁇ M fenofibrate (A), Also shown are the results (B) of analysis of the neutral fat (triglyceride) content of large, medium, and small sizes of VLDL in the culture supernatant.
  • the human fatty liver model cells of the present invention can be produced by a method for producing human fatty liver model cells comprising the step of culturing fatty liver-derived human hepatocytes in a medium containing dimethylsulfoxide. can.
  • the method for producing human fatty liver model cells of the present invention is described below.
  • Fatty liver-derived human hepatocytes refers to human hepatocytes collected from fatty liver tissue, or those that have been frozen and then thawed. can be used.
  • Fatty liver tissue is a fatty liver tissue derived from a human patient with fatty liver, a non-human animal model obtained by transplanting human liver cells into an immunodeficient non-human animal with liver disease (hereinafter referred to as "chimeric non-human animal”). described) can be used.
  • Human hepatocytes can be collected from fatty liver tissue by conventionally known methods, namely collagenase perfusion method, centrifugation, elutriator, FCM (flow cytometry), means such as monoclonal antibodies that specifically recognize human hepatocytes. can be done using
  • the “fatty liver-derived human hepatocytes” are preferably human hepatocytes recovered from chimeric non-human animals from the viewpoint of being able to be mass-produced and stably supplied.
  • Human hepatocytes recovered from chimeric non-human animals Human liver cells recovered from fatty liver tissue derived from chimeric non-human animals that can be used in the present invention can be prepared by the following method.
  • chimeric non-human animal means a non-human animal in which part or all of the hepatocytes in the liver are replaced with human hepatocytes.
  • Non-human animals are preferably mammals, more preferably rodents. Rodents include rodents such as mice and rats, guinea pigs, squirrels, and hamsters, and rats such as mice and rats, which are widely used as laboratory animals, are particularly preferred.
  • Chimeric non-human animals having human hepatocytes are obtained by transplanting human hepatocytes into liver-damaged and immunodeficient non-human animals according to conventionally known techniques (Japanese Unexamined Patent Application Publication No. 2002-45087, WO2008/001614, WO2013/145331). can be obtained by
  • Hepatic-damaged and immunodeficient non-human animals refer to immunodeficient animals that do not show rejection against heterologous animal-derived cells and means an animal in which the cells of the cell are damaged. Since the non-human animal-derived liver cells are damaged, the transplanted human hepatocytes easily proliferate, and the transplanted human hepatocytes maintain liver function.
  • liver-damaged and immunodeficient non-human animal can be produced by subjecting the same individual to liver damage-inducing treatment and immunodeficiency-inducing treatment.
  • liver injury-inducing treatment include administration of liver injury-inducing substances (e.g., carbon tetrachloride, yellow phosphorus, D-galactosamine, 2-acetylaminofluorene, pyrrolodin alkaloids, etc.) and surgical treatment (e.g., liver injury). partial excision, etc.).
  • liver injury-inducing substances e.g., carbon tetrachloride, yellow phosphorus, D-galactosamine, 2-acetylaminofluorene, pyrrolodin alkaloids, etc.
  • surgical treatment e.g., liver injury). partial excision, etc.
  • immunodeficiency-inducing treatment include administration of an immunosuppressive agent and removal of the thymus.
  • a liver-damaged immunodeficient non-human animal can be produced by subjecting a genetically immunodeficient animal to liver damage-inducing treatment.
  • genetically immunodeficient animals include animals with severe combined immunodeficiency (SCID) showing T-cell lineage deficiency, animals with lost T-cell function due to genetic thymic deficiency, and animals with the RAG2 gene. Knockout animals and the like can be used.
  • SCID mice NUDE mice, RAG2 knockout mice, IL2Rgc/Rag2 knockout mice, NOD mice, NOG mice, nude mice, nude rats, X-ray irradiated nude rats obtained by transplanting bone marrow of SCID mice Immunodeficient rats (Japanese Unexamined Patent Application Publication No. 2007-228962, Transplantation, 60(7):740-7, 1995) and the like can be used.
  • a liver-damaged, immunodeficient non-human animal can also be produced by subjecting an animal with genetic liver damage to an immunodeficiency-inducing treatment.
  • animals with genetic liver damage transgenic animals into which a liver damage-inducing protein gene linked under the control of an enhancer and/or promoter of a protein specifically expressed in hepatocytes has been introduced can be used.
  • Hepatocyte-specific protein includes serum albumin, cholinesterase, Hagemann factor, etc. Enhancers and/or promoters that regulate the expression of these genes can be used.
  • Hepatopathy-inducing proteins include urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), and the like.
  • liver injury is induced because a liver injury-inducing protein is expressed in a hepatocyte-specific manner under the enhancer and/or promoter of a protein specifically expressed in hepatocytes.
  • animals with genetic liver disorders can be produced by knocking out genes responsible for liver function. Examples of the "gene responsible for liver function" include fumarylacetoacetate hydrolase gene and the like.
  • a liver-damaged, immunodeficient non-human animal can be produced by mating a genetically immunodeficient animal with a genetically liver-damaged animal of the same species.
  • liver injury immunodeficient non-human animals gene targeting, CRISPR-Cas9, zinc finger nuclease (ZFN), TALE nuclease (TALEN) and other genome editing technology and genetic recombination technology
  • the above immunity Fertilized eggs and pluripotent stem cells derived from non-human animals, or non-human animals that are genetically immunodeficient and/or have genetic liver damage, as genetic factors that cause insufficiency and/or liver damage can be produced by introducing (Wang, H. et al., Cell, 153, 910-918, (2013) Yang, H.
  • the "liver-damaged immunodeficient non-human animal” may have a gene that defines the trait of immunodeficiency and a gene that defines the trait of liver damage, each of which is homozygous, or heterozygous. may have
  • the liver-damaged and immunodeficient non-human animals of the present invention are represented by genotypes such as uPA (+/-)/SCID (+/+) and uPA (+/+)/SCID (+/+). Liver-damaged immunodeficient mice can be preferably used.
  • the "human hepatocytes" to be transplanted into the liver-damaged immunodeficient non-human animal may be human-derived hepatocytes.
  • Human hepatocytes isolated according to conventional methods such as the collagenase perfusion method can be used.
  • the human liver tissue may be liver tissue derived from a healthy individual, or may be liver tissue derived from a patient suffering from a disease such as fatty liver or liver cancer, but is preferably liver tissue derived from a healthy individual. be.
  • the age of the human from which hepatocytes are to be isolated is not particularly limited, it is preferable to isolate from the liver tissue of children under the age of 14. By using hepatocytes from children under the age of 14, a high replacement rate with human hepatocytes can be achieved after transplantation.
  • the isolated hepatocytes can be frozen and stored once and then thawed before use.
  • human hepatocytes may be proliferative human hepatocytes that have the ability to actively proliferate in vivo.
  • proliferating human hepatocytes means human hepatocytes that form colonies as populations of a single cell type under culture conditions (in vitro) and proliferate to increase the colonies.
  • the expansion is sometimes referred to as "clonal expansion" in that the colony-constituting cells are of a single species.
  • such cells are cells whose cell number can be further increased by subculturing.
  • proliferative human hepatocytes examples include human small hepatocytes (Japanese Patent Application Laid-Open No. 08-112092; Japanese Patent No. 3266766; US Patent No. 6,004,810; Japanese Patent Application Laid-Open No. 10-179148: Japanese Patent No. 3211941, Japanese Unexamined Patent Publication No. 7-274951; Japanese Patent No. 3157984, Japanese Unexamined Patent Publication No. 9-313172; Japanese Patent No. 3014322).
  • the isolated human hepatocytes may be used as they are or after further purification. Purification of hepatocytes can be performed according to a conventional method, for example, using means such as centrifugation, elutriator, FCM, and a monoclonal antibody that specifically recognizes hepatocytes proliferating while forming colonies. can.
  • a monoclonal antibody that specifically recognizes hepatocytes proliferating while forming colonies.
  • Known monoclonal antibodies (WO2008/001614) that specifically recognize human hepatocytes and proliferating human hepatocytes can be used.
  • the human hepatocytes are human hepatocytes isolated from the liver tissue of a chimeric non-human animal having human hepatocytes according to a conventional method such as the collagenase perfusion method, or the human hepatocytes once frozen and then thawed.
  • human hepatocytes obtained by inducing pluripotent stem cells (e.g., embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), etc.), hepatic progenitor cells such as Clip cells, in vitro Proliferated human hepatocytes, cryopreserved hepatocytes, hepatocytes immortalized by introduction of a telomerase gene or the like, and mixtures of these hepatocytes and non-parenchymal cells can also be used.
  • pluripotent stem cells e.g., embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), etc.
  • hepatic progenitor cells such as Clip cells
  • in vitro Proliferated human hepatocytes in vitro Proliferated human hepatocytes
  • cryopreserved hepatocytes hepatocytes immortalized by introduction of a telomerase gene or the like
  • Transplantation of human hepatocytes into liver-damaged and immunodeficient non-human animals can be performed by transplanting into the liver of the non-human animal via the spleen. Alternatively, it can be implanted directly through the portal vein.
  • the number of human hepatocytes to be transplanted can be about 1 to 2 million, preferably about 200,000 to 1,000,000.
  • the sex of the liver-damaged immunodeficient non-human animal is not particularly limited.
  • the age of liver-damaged, immunodeficient non-human animals used for transplantation is not particularly limited, but if human hepatocytes are transplanted at a low age, the human hepatocytes can proliferate more actively as the animal grows. 0 to 40 days after birth, preferably about 8 to 40 days after birth can be used.
  • Animals after transplantation can be bred by conventional methods. For example, by rearing for about 40 to 200 days after transplantation, a chimeric non-human animal in which some or all of the hepatocytes in the non-human animal have been replaced with human hepatocytes can be obtained. In the liver of the chimeric non-human animal thus obtained, symptoms of fatty liver such as large lipid droplets and liver steatosis are observed (WO2008/001614).
  • Human hepatocytes can be collected from the chimeric non-human animal according to a conventional method such as the collagenase perfusion method.
  • the collection of human hepatocytes is preferably performed using a chimeric non-human animal in which human hepatocytes are contained at a high rate in the collected hepatocytes.
  • a chimeric non-human animal having one or more of the following characteristics It can be done using animals.
  • hepatocytes in the liver are human hepatocytes is replaced by (ii) a blood human albumin level of 0.1 mg/mL or more, preferably 0.5 mg/mL or more, more preferably 1 mg/mL or more, even more preferably 5 mg/mL or more, and even more preferably 10 mg/mL or more; be; (iii) 12 to 21 weeks, preferably 13 to 20 weeks, more preferably 14 to 19 weeks have passed since the transplantation of human hepatocytes.
  • the collected human hepatocytes may be used as they are, or human hepatocytes may be purified using a monoclonal antibody that specifically recognizes human hepatocytes or non-human animal hepatocytes.
  • a monoclonal antibody that specifically recognizes human hepatocytes or non-human animal hepatocytes.
  • the separated hepatocytes are reacted with a human hepatocyte-specific monoclonal antibody
  • the cells bound to the antibody are collected by FCM or a magnetic cell separation device, and the separated hepatocytes are treated as non-human animal hepatocytes.
  • human hepatocytes can be purified and collected by collecting cells that do not bind to the antibody using FCM or a magnetic cell separation device.
  • the collected human hepatocytes may be further collected in the same manner after being transplanted into a new liver-damaged, immunodeficient non-human animal as described above (passive transplantation). Passaging can be performed once or multiple times (eg, 2-4 times).
  • fatty liver-derived human hepatocytes can be cultured using a medium generally used for culturing animal cells.
  • a medium generally used for culturing animal cells.
  • a medium include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), Williams Medium E, and the like.
  • DMEM Dulbecco's Modified Eagle Medium
  • Fetal bovine serum, insulin, epidermal growth factor, dexamethasone, buffers, antibiotics, pH adjusters, proline, ascorbic acid, nicotinamide and the like can be added to the medium as necessary.
  • DMSO dimethyl sulfoxide
  • DMSO can be added to the medium in an amount (final concentration) of 1 to 4% by weight, preferably 1 to 2% by weight, for example 2% by weight.
  • Fatty liver-derived human hepatocytes are added to the medium at 0.21-21.3 ⁇ 10 5 cells/cm 2 , preferably 1.07-3.2 ⁇ 10 5 cells/cm 2 , for example 2.13. Seed at a volume of x105 cells/ cm2 . If the number of cells is less than 0.21, it may not be possible to obtain a sufficient amount of cells for human fatty liver model cells, while if the number of cells is more than 21.3 ⁇ 10 5 , cell growth will decrease and lipid content will decrease. Decrease in secretion and/or accumulation, etc. may occur.
  • Fatty liver-derived human hepatocytes may be cultured for a period of time sufficient for the cells to secrete and/or accumulate lipids, and are not particularly limited. 2-fold or more, 3-fold or more, 4-fold or more, or 5-fold or more, preferably 6-fold or more, more preferably 7-fold or more, more preferably 8-fold or more, than human hepatocytes derived from fatty liver cultured under conditions, Even more preferably, 9 times more lipid droplets can be formed for a sufficient period of time for cells to be encapsulated.
  • Such a culture period can be, for example, longer than 3 days, preferably 4 days or longer, and more preferably 5 days or longer.
  • the upper limit of the culture period is not particularly limited, it can be, for example, 17 days or less, preferably 13 days or less.
  • the medium can be replaced as appropriate during the culture period.
  • Human hepatocytes that secrete and/or accumulate lipids after the end of culture can be used as human fatty liver model cells.
  • the human fatty liver model cells of the present invention encapsulate many lipid droplets and secrete and / or accumulate a lot of lipids, similar to hepatocytes in human fatty liver. It is a human-derived cultured hepatocyte in which the expression of fatty liver-related genes and the like is observed and maintains the symptoms of fatty liver, more specifically, a monolayer hepatocyte culture.
  • the human fatty liver model cells of the present invention enclose many lipid droplets and have a high lipoprotein content and/or secretion amount.
  • "encapsulating many lipid droplets” means that in the culture of the above-mentioned fatty liver-derived human hepatocytes, cultured under the same conditions in the same medium except that DMSO was not added (e.g., more than 3 days 2 times or more, 3 times or more, 4 times or more, or 5 times or more, preferably 6 times or more, than fatty liver-derived human hepatocytes cultured for a long time, preferably 4 days or more, more preferably 5 days or more, It means that the human fatty liver model cells of the present invention encapsulate lipid droplets that are more preferably 7 times or more, still more preferably 8 times or more, still more preferably 9 times or more.
  • Lipoprotein is a complex particle for transporting lipids such as cholesterol and triglycerides from the site of absorption/synthesis to the site of use. It has a structure in which free cholesterol and apolipoprotein are arranged, and hydrophobic cholesterol and neutral lipid are arranged inside the particle.
  • High lipoprotein content and/or secretion amount means that in the culture of human hepatocytes derived from fatty liver described above, cultured under the same conditions in the same medium except that DMSO was not added (e.g., Longer than 3 days, preferably 4 days or more, more preferably 5 days or more cultured) human hepatocytes derived from fatty liver or lipoproteins (more preferably triglycerides) contained in the culture supernatant, 5 More than twice, preferably more than 6 times, more preferably more than 7 times, more preferably more than 8 times, even more preferably more than 9 times the amount of lipoprotein is the human fatty liver model cell of the present invention or its culture supernatant means contained within.
  • the amount of lipoproteins in cells or culture supernatant can be measured by a conventionally known technique as described below.
  • the human fatty liver model cell of the present invention can also be characterized by the lipoprotein subclass content in the culture supernatant.
  • Lipoproteins can be classified into several subclasses depending on their properties such as particle size, hydration density, and electrophoretic mobility.
  • lipoproteins are classified into chylomicrons (CM), ultra-low specific gravity Lipoprotein (Very Low Density Lipoprotein; VLDL), Low Density Lipoprotein (LDL), and High Density Lipoprotein (HDL) can be roughly classified into four.
  • VLDL has three further subclasses Large, Medium, and Small
  • LDL has four further subclasses Large, Medium, Small, and Very Small
  • HDL has five further subclasses Very Large, Large, Medium, Small, and Very Small.
  • Subclasses can be classified, each based on particle size.
  • CM has two further subclasses G01, G02
  • VLDL has five further subclasses G03, G04, G05, G06, G07
  • LDL has six further subclasses G08, G09, G10, G11, G12, G13.
  • HDL can be divided into seven further subclasses, G14, G15, G16, G17, G18, G19 and G20, each based on particle size.
  • Each subclass of lipoproteins in the culture supernatant was analyzed by a conventionally known method using gel filtration liquid chromatography (WO2007/052789; JP-A-9-15225; Arterioscler Thromb Vasc Biol. 2005; 25: 1-8 ; LipoSEARCH (registered trademark) (Skylight Biotech Co., Ltd.).
  • VLDL exhibits the highest content among lipoproteins.
  • VLDL shows a higher content than LDL, for example, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times 8-fold or more, 9-fold or more, 10-fold or more, or 15-fold or more amounts are included.
  • the content of VLDL cholesterol is higher than that of LDL cholesterol.
  • 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more, or 15 times or more contains an amount of
  • the content of VLDL is higher than that of HDL. Included are amounts of 30-fold or more, 40-fold or more, 50-fold or more, 60-fold or more, 70-fold or more, 80-fold or more, or 90-fold or more.
  • the content of VLDL cholesterol is higher than that of HDL cholesterol. More than twice, more than 25 times, more than 30 times, more than 40 times, more than 50 times, more than 60 times, more than 70 times, more than 80 times, or more than 90 times the amount of VLDL triglycerides Shows a higher content than neutral fat (triglyceride) of HDL, for example, 5 times or more, 10 times or more, 15 times or more, 20 times or more, 25 times or more, 30 times or more, 40 times or more, 50 times or more, Included are amounts of 60-fold or greater, 70-fold or greater, 80-fold or greater, or 90-fold or greater.
  • LDL shows a higher content than HDL, for example, 2.5 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times and greater, 7-fold or greater, 8-fold or greater, 9-fold or greater, or 10-fold or greater amounts are included.
  • the content of LDL cholesterol is higher than that of HDL cholesterol, for example, 2.5 times or more, 3 times or more, 4 times or more. , 5 times more, 6 times more, 7 times more, 8 times more, 9 times more, or 10 times more triglycerides in LDL than triglycerides in HDL showing a high content, e.g. be
  • Large-VLDL is contained in the highest ratio, and 70 masses of the entire VLDL % or more, preferably 75% by mass or more, more preferably 80% by mass or more, still more preferably 81% by mass or more, even more preferably 82% by mass or more (e.g., 83% by mass or more, 84% by mass or more). It is contained at a rate of 85% by mass or more.
  • VLDL neutral fat is large-VLDL, medium-VLDL, and small-VLDL neutral fat (triglycerides)
  • Large-VLDL neutral fat is contained in the highest proportion, 70% by mass or more, preferably 75% by mass or more, more preferably 75% by mass or more of the total amount of VLDL neutral fat (triglyceride) 80% by mass or more, more preferably 81% by mass or more, still more preferably 82% by mass or more (for example, 83% by mass or more, 84% by mass or more), and particularly preferably 85% by mass or more.
  • VLDL cholesterol is the sum of Large-VLDL, Medium-VLDL, and Small-VLDL cholesterols, of which Large-VLDL cholesterol is the highest. It is contained in a high proportion and is 70% by mass or more, preferably 75% by mass or more, more preferably 80% by mass or more, still more preferably 81% by mass or more, and even more preferably 82% by mass or more of the total amount of VLDL cholesterol (e.g. , 83% by mass or more, 84% by mass or more) is particularly preferably contained in a proportion of 85% by mass or more.
  • the human fatty liver model cell of the present invention can also be characterized by the expression level of the fatty liver-related gene in the cell.
  • fatty liver-related gene means a gene whose expression level is increased in hepatocytes of fatty liver compared to hepatocytes of healthy liver.
  • fatty liver-related genes include a gene encoding fatty acid synthase (gene name: FASN), a gene encoding SREBP-1 (gene name: SREBF1), and a gene encoding glucose-6-phosphatase (G6PC).
  • the gene encoding cholesterol 7 ⁇ -hydroxylase (CYP7A1) the gene encoding cholesteryl ester transfer protein (CETP), the gene encoding glucokinase (GCK), the gene encoding phosphoenolpyruvate carboxykinase 1 (PCK1) etc.
  • the "high" expression level of the fatty liver-related gene is the above-mentioned fatty liver-derived human liver cultured in a DMSO-free medium or cultured in a DMSO-containing medium for 3 days or less It means that it is higher than the expression level of a fatty liver-related gene in cells, for example, 2-fold or more, preferably 3-fold or more, more preferably 3.5-fold or more.
  • the expression level of a fatty liver-related gene can be quantified by a conventionally known method, preferably by microarray analysis.
  • the human fatty liver model cells of the present invention are preferably cultured in a medium containing DMSO.
  • the human fatty liver model cells of the present invention show the highest content of VLDL among lipoproteins, compared to known hepatocytes (HepG2, HuH7, etc.), the model is more similar to human fatty liver hepatocytes. can be used as
  • the human fatty liver model cells of the present invention can be used as a model of human fatty liver, and can be used to detect changes in the very low density lipoprotein (VLDL) content in the culture supernatant. As an index, it can be used in a method of screening for substances that are effective against dyslipidemia including fatty liver.
  • VLDL very low density lipoprotein
  • Screening is carried out by administering a test substance to the culture of human fatty liver model cells of the present invention, and determining whether or not VLDL in the culture supernatant increases or decreases between the cells to which the test substance has been administered and the cells to which the test substance has not been administered, and/or Alternatively, it can be performed by comparing the degree of increase or decrease. More specifically, the screening involves administering a test substance to the culture of human fatty liver model cells of the present invention, and dividing VLDL in the culture supernatant between cells to which the test substance has been administered and cells to which it has not been administered. This can be done by comparing the presence or absence of an increase or decrease in the amount of sexual fat (triglyceride) or cholesterol and/or the degree of increase or decrease.
  • sexual fat triglyceride
  • the screening is performed by administering a test substance to the culture of the human fatty liver model cell of the present invention, and comparing Large-VLDL in the culture supernatant between the cells to which the test substance was administered and the cells to which the test substance was not administered. It can be performed by comparing the presence or absence of increase/decrease and/or the degree of increase/decrease. More specifically, the screening is performed by administering a test substance to the culture of human fatty liver model cells of the present invention, and detecting Large-VLDL in the culture supernatant between cells to which the test substance has been administered and cells to which it has not been administered.
  • Test substance-administered cells and non-administered cells may be the same culture before and after administration of the test substance, or separate cultures that are manipulated in the same manner except for the presence or absence of test substance administration. It may be a culture.
  • dyslipidemia includes fatty liver, dyslipidemia (hyperlipidemia), and diseases and symptoms caused by or at a high risk of being caused by them (e.g., arteriosclerosis, thrombosis, NASH, cirrhosis, liver cancer , myocardial infarction, stroke, etc.).
  • “substances having an effect on dyslipidemia” include substances that are effective for dyslipidemia, substances that adversely affect dyslipidemia, and the like.
  • a “substance effective against dyslipidemia” means a substance capable of reducing the amount of VLDL produced in human fatty liver cells. More specifically, the substance means a substance capable of reducing the amount of triglycerides or cholesterol in VLDL produced by human fatty liver cells. More preferably, the substance means a substance capable of reducing the amount of Large-VLDL secreted in human fatty liver cells. More specifically, the substance means a substance capable of lowering the amount of triglycerides or cholesterol in Large-VLDL produced by human fatty liver cells. These substances may contribute to treating or ameliorating dyslipidemia.
  • the amount of VLDL (especially, the amount of VLDL neutral fat (triglyceride) or cholesterol), preferably the amount of Large-VLDL (especially, When a decrease in neutral fat (triglyceride amount) or cholesterol amount) in Large-VLDL is confirmed, the test substance improves at least one symptom of dyslipidemia, especially fatty liver, and the present invention It can be determined to be a "substance effective against dyslipidemia".
  • Large-VLDL produces Small-LDL through metabolism (Ronald M Krauss, Diabetes Care.
  • the amount of VLDL (especially, the amount of VLDL neutral fat (triglyceride) or cholesterol), preferably the amount of Large-VLDL (especially, When a decrease in triglyceride (triglyceride) content or cholesterol content in Large-VLDL is confirmed, the test substance improves at least one symptom of dyslipidemia, particularly arteriosclerosis. It can be determined to be a "substance effective against dyslipidemia". Specific examples of substances that cause such a decrease in the amount of VLDL include fenofibrate, lomitapide, etc., which are known as active ingredients of antihyperlipidemia drugs.
  • the human fatty liver model cells of the present invention can be used for detection using the increase or decrease of VLDL in the cells as an index.
  • Substances that are effective against dyslipidemia diseases and symptoms can be determined to be effective not only in the treatment or improvement of the diseases and symptoms, but also in the prevention thereof.
  • a substance that adversely affects dyslipidemia means a substance that can exacerbate at least one symptom in human fatty liver cells.
  • the substance is the amount of VLDL produced by human fatty liver cells (especially the amount of neutral fat (triglyceride) or cholesterol in VLDL), preferably the amount of Large-VLDL (especially the amount of Large-VLDL It means a substance capable of increasing neutral fat (triglyceride content) or cholesterol content).
  • lipid abnormalities such as fatty liver and arteriosclerosis, VLDL content (especially VLDL neutral fat (triglyceride) content or cholesterol content), preferably Large-VLDL content (especially Large-VLDL neutral fat content) (triglyceride content) or cholesterol content).
  • the amount of VLDL (especially, the amount of VLDL neutral fat (triglyceride) or cholesterol), preferably the amount of Large-VLDL (especially, If an increase in the neutral fat (triglyceride) content or cholesterol content of Large-VLDL is confirmed, the test substance can exacerbate at least one symptom of dyslipidemia such as fatty liver or arteriosclerosis. , can be determined to be the "substance that adversely affects dyslipidemia" of the present invention. Specific examples of substances that cause such an increase in the amount of VLDL include oleic acid and the like. It can be detected using human fatty liver model cells.
  • Test substances include, but are not limited to, low-molecular-weight compounds, amino acids, nucleic acids, lipids, sugars, extracts of natural products, and the like.
  • PXB mice production of chimeric mice (PXB mice) having human hepatocytes)
  • PXB mice were produced according to a conventionally known method (Japanese Patent Application Laid-Open No. 2002-45087). Namely, the enhancer of albumin synthesized in the liver and the urokinase plauminogen activator (uPA) gene (cDNA-uPA) linked to the promoter were introduced into all cells, and immunodeficient mice (SCID) were introduced into all cells. mice) were mated to produce immunodeficient liver-damaged mice (cDNA-uPA(+/-)/SCID mice).
  • uPA urokinase plauminogen activator
  • a 3-week-old cDNA-uPA(+/-)/SCID mouse was anesthetized, the skin around the spleen and the rectus abdominis muscle were incised with scissors, and the tip of the spleen was pinched to fix it at a position where cells could be easily injected. Then, a glass syringe filled with a human hepatocyte suspension was used to inject the cells by pricking the tip of the spleen.
  • the spleen was put back into the body of the mouse, the skin and peritoneum were sewn up with a plastic surgical needle, the incision was closed, and the mouse to be transplanted was confirmed to have no abnormality in respiration, and was reared in a breeding cage.
  • PXB mice that are 17 to 22 weeks old, weigh 15 to 20 g, and have a serum human albumin level of 10 mg/mL or higher (human hepatocyte replacement rate of 95% or higher calculated from the human albumin level) were selected. , were used in the following experiments.
  • the dilution factor for the desired seeding density was calculated and diluted with medium A. Gently pour 500 ⁇ L of the diluted cell suspension into each well of the culture plate, allow to stand for about 20 minutes until the cells lightly adhere to the bottom, then gently transfer to an incubator (37° C., 5% CO 2 ) and culture. did.
  • DMSO-containing medium On the day after seeding, medium A was removed, 500 ⁇ L of medium B (DMSO(+)) was added, the cells were gently transferred to an incubator (37°C, 5% CO 2 ), and cultured. . On the 5th, 7th, 8th, and 12th days from the start of culture using medium B (DMSO(+)), the medium was replaced with new medium B (DMSO(+)). After 14 days of culture using medium B (DMSO(+)), the intracellular cholesterol and triglyceride levels were analyzed for lipoproteins in the cells as described below.
  • HepG2 cells, HuH7 cells Culture of Known Hepatocytes
  • Known hepatocytes were seeded in 500 ⁇ L of medium A, gently transferred to an incubator (37° C., 5% CO 2 ), and cultured for 7 days. After completion of the culture, analysis of lipoproteins in the culture supernatant described below and analysis of lipoproteins in the cells described below were performed.
  • the medium used for culture was removed, 500 ⁇ L of medium D for analysis was added, the cells were gently transferred to an incubator (37° C., 5% CO 2 ), and cultured for 2 days. After that, the culture supernatant was collected, and the lipoproteins contained in the culture supernatant (80 ⁇ L) were fractionated into four subgroups, CM, VLDL, LDL, and HDL fractions, by gel filtration HPLC. Cholesterol and triglycerides contained in each fraction were quantified by online enzymatic reaction. Concentration analysis was performed using a computer program unique to Skylight Biotech.
  • Diacolor Liquid TG-S (manufactured by Toyobo) was used, and standard serum for cholesterol and triglyceride concentrations in CM, VLDL, LDL, and HDL was manufactured by Kyowa Hakko Kirin Co. It was used.
  • Triglycerides in cells were measured using Cholestest (registered trademark) TG (Sekisui Medical Co., Ltd.) according to the manufacturer's instructions. That is, after the cells were washed with PBS, the water was completely drained (stored at -80°C until measurement), 200 ⁇ L of TG enzyme solution (1) was added to each cell well, and the mixture was incubated at 37°C for 10 minutes to react. (remove free glycerol). Next, detach the cells with a pipette, transfer to a centrifuge tube and centrifuge at 10,000 rpm for 10 minutes.
  • Cholestest registered trademark
  • TG Sekisui Medical Co., Ltd.
  • Cholesterol in cells was measured using Cholestest (registered trademark) CHO (Sekisui Medical Co., Ltd.) according to the manufacturer's instructions. That is, after the cells were washed with PBS, the water was completely drained (stored at -80°C until measurement), 200 ⁇ L of CHO enzyme solution (1) was added to each cell well, and the cells were incubated at 37°C for 10 minutes.
  • RNA from PXB-cells cultured in medium B for 3 days or 6 days was extracted using TRIzol (registered trademark) + Direct zol (Thermo Fisher Scientific) according to the manufacturer's instructions.
  • microarray analysis was performed according to the manufacturer's instructions, and fatty liver-related genes FASN, SREBF1, The expression levels of G6PC, CYP7A1, CETP, GCK, and PCK1 were analyzed.
  • Perfusate and medium Perfusate A, perfusate B, perfusate C, medium A, medium B, medium C, and medium D had the following compositions.
  • FIG. 1 shows PXB-cells cultured for 5 days in medium B (DMSO(+)) or medium C (DMSO(-)).
  • medium B DMSO (+)
  • DMSO (+) DMSO (+)
  • DMSO (+) DMSO (+)
  • DMSO (-) medium C
  • lipid droplets shown in white
  • FIG. 2 shows lipoproteins (including CM, VLDL, LDL, and HDL) in the culture supernatant collected after 5 days of culture in medium B (DMSO (+)) or medium C (DMSO (-)). shows the measurement results of the total neutral fat (triglyceride) content of The results are shown as relative values with the content in the culture supernatant of PXB-cells cultured in medium B (DMSO(+)) being "100". PXB-cells cultured in medium B (DMSO(+)) secreted higher (approximately 9-fold) total triglycerides in lipoproteins than cultured in medium C (DMSO(-)) was accepted.
  • DMSO (+) DMSO (+)
  • DMSO (-) medium C
  • Fig. 3 shows PXB-cells cultured for 5, 9, 12, and 14 days in medium B (DMSO (+)), and The analysis results of lipoproteins in each culture supernatant of known hepatocytes (HepG2 cells, HuH7 cells) are shown.
  • FIG. 4 shows the results of analyzing four subgroups of lipoproteins contained in the culture supernatant.
  • PXB-cells cultured in medium B (DMSO(+)) were confirmed to contain the most VLDL in both cholesterol and triglycerides, regardless of the culture period.
  • DMSO (+) medium B (DMSO (+)) (containing CM, VLDL, LDL, HDL ratio (weight ratio)).
  • VLDL peak observed in PXB-cells was not observed in the culture supernatants of HepG2 cells and HuH7 cells, and was specifically secreted in PXB-cells cultured in medium B (DMSO (+)). It was confirmed that
  • Table 5 shows the results of analysis of the amount of neutral fat (triglyceride) in Large-VLDL, Medium-VLDL, and Small-VLDL with respect to VLDL contained in the culture supernatant.
  • PXB-cells were cultured in medium B (DMSO(+)) for 13 days and then cultured in medium D for 2 days; Fresh human hepatocytes (BIOPREDIC International) (FHH) isolated from a healthy subject (no history of disease) cultured for 2 days; and HepG2 cells cultured in medium A for 2 days and then in medium D for 2 days, and HuH7 cells; and the analysis results of each culture supernatant.
  • medium B DMSO(+)
  • FHH Fresh human hepatocytes isolated from a healthy subject (no history of disease) cultured for 2 days
  • HepG2 cells cultured in medium A for 2 days and then in medium D for 2 days, and HuH7 cells
  • the ratio of Large-VLDL to the total VLDL in the culture supernatant is 81.5% (40.9/50.2) for PXB-cells, 69.2% (78.6/113.5) for FHH, It was 0% in HepG2 cells and 1.9% in HuH7 cells.
  • PXB-cells cultured in medium B (DMSO(+)) showed a significantly high proportion of Large-VLDL content.
  • PXB-cells cultured for 5, 9, 12, and 14 days in medium B (DMSO (+)), and known liver The analysis results of lipoproteins in each cell (HepG2 cells, HuH7 cells) are shown.
  • Fatty liver-related gene expression level Fig. 6 shows the result of analyzing the expression level of fatty liver-related genes (FASN, SREBF1, G6PC) in PXB-cells cultured for 3 days and 6 days in medium B (DMSO (+)) indicates The results are shown as relative values where the expression level of each gene on day 3 of culture is set to "1". For all genes, an increase in expression level was observed from day 3 to day 6 of culture. Moreover, although not shown in FIG. 6, an increase in the expression level of CYP7A1, CETP, GCK, and PCK1 was similarly observed from day 3 to day 6 of culture.
  • PXB-cells cultured in medium B can maintain the accumulation and secretion of lipids, and are also accompanied by the expression of fatty liver-related genes. It was confirmed that it can be used as a model cell.
  • oleic acid which is known to promote lipid accumulation in hepatocytes
  • the PXB-cells medium prepared above was replaced with medium D containing 0.75 mM oleic acid, gently transferred to an incubator (37° C., 5% CO 2 ), and cultured for 2 days. After completion of the culture, the total cholesterol and total triglycerides in the culture supernatant and cells were analyzed by the methods described in "Analysis of lipoproteins in culture supernatant" and "Analysis of lipoproteins in cells” above. ) were measured respectively. For controls, medium D (5 ⁇ L) alone was added instead of oleic acid.
  • lomitapide (Tokyo Kasei Co., Ltd.), which is known as a hyperlipidemia drug, was used.
  • the PXB-cells medium prepared above was replaced with medium D containing 1 nM and 5 nM lomitapide, respectively, and gently transferred to an incubator (37° C., 5% CO 2 ) and cultured for 2 days.
  • cholesterol and triglycerides in the culture supernatant and cells were removed by the methods described in the above "Analysis of lipoproteins in culture supernatant" and "Analysis of lipoproteins in cells”. measured respectively.
  • ethanol 5 ⁇ L was added instead of lomitapide.
  • FIG. 8 shows the result of analyzing the neutral fat (triglyceride) content in each subgroup of lipoproteins in the culture supernatant of PXB-cells cultured for 2 days with the addition of lomitapide (A ), and the result (B) of analyzing the neutral fat (triglyceride) content of each size of Large, Medium, and Small for VLDL of the culture supernatant.
  • the PXB-cells medium prepared above was replaced with medium D containing 10 ⁇ M, 50 ⁇ M and 100 ⁇ M of fenofibrate, respectively, gently transferred to an incubator (37° C., 5% CO 2 ), and cultured for 2 days. After completion of the culture, cholesterol and triglycerides in the culture supernatant and cells were removed by the methods described in the above "Analysis of lipoproteins in culture supernatant" and "Analysis of lipoproteins in cells”. measured respectively. For controls, only ethanol (5 ⁇ L) was added instead of fenofibrate.
  • FIG. 9 shows the result of analyzing the neutral fat (triglyceride) content in each subgroup of lipoproteins in the culture supernatant of PXB-cells cultured for 2 days with the addition of fenofibrate.
  • A and the result (B) of analyzing the neutral fat (triglyceride) content of each size of Large, Medium, and Small for VLDL of the culture supernatant.
  • the amount of VLDL, especially Large-VLDL, in the culture supernatant (especially its neutral fat (triglyceride ) amount) as an indicator, it can be used to prevent, treat, or improve lipid abnormalities, including substances that adversely affect lipid abnormalities (oleic acid, etc.), lipid metabolism improving agents, and hyperlipidemia drugs. It was confirmed that effective substances (lomitapide, fenofibrate, etc.) can be screened.

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