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WO1998058050A1 - Lignee cellulaire hepatique humaine immortalisee et procede de preparation associe - Google Patents

Lignee cellulaire hepatique humaine immortalisee et procede de preparation associe Download PDF

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WO1998058050A1
WO1998058050A1 PCT/US1997/019764 US9719764W WO9858050A1 WO 1998058050 A1 WO1998058050 A1 WO 1998058050A1 US 9719764 W US9719764 W US 9719764W WO 9858050 A1 WO9858050 A1 WO 9858050A1
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Prior art keywords
cells
cell line
human
hepatic
liver
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Judith F. H. Blasser
Chia Chiao
William K. Kaufmann
David G. Kaufman
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University of North Carolina at Chapel Hill
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University of North Carolina at Chapel Hill
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/80—Undefined extracts from animals
    • C12N2500/84—Undefined extracts from animals from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/04—Immortalised cells

Definitions

  • This application relates to immortalized cell lines, and more specifically to immortalized, human hepatic cells.
  • T-antigen has been shown to associate with tumor suppressor genes. See DeCaprio et al . , Cell 54, 275-83 (1988) . This association may add undesirable changes to the cellular characteristics including karyotype instability, de-differentiation, and transformation over repeated passaging.
  • JB-HEPl is immortal, yet preserves morphological features of differentiated hepatocytes and retains major regulatory pathways that control hepatocyte gene expression and differentiation.
  • one aspect of the present invention is a method of providing an immortalized human hepatic cell line, comprising the steps of collecting normal hepatic cells from human liver; transforming said normal hepatic cells with at least one exogenous gene; and maintaining said cells in serum-free media.
  • FIG. 1A is a light micrograph showing the morphology of a cell taken from the JB-HEPl cell line in monolayer culture, and illustrating the hepatocyte cordlike structure in non-confluent cells.
  • FIG. IB is a light micrograph showing the morphology of the JB-HEPl cell line in monolayer, confluent culture.
  • FIG. IC is an oil-immersion light micrograph showing detailed hepatocyte structure of the JB-HEPl cell line in culture.
  • FIG. 2 is a karyotype of a cell of the JB-HEPl cell line at the 90th passage.
  • the chromosome complement is near tetraploid with several derivative chromosomes .
  • FIG. 3A is a transmission electron micrograph of the JB-HEPl cell line at the 90th passage, illustrating the ultrastructure of the cells in monolayer culture.
  • FIG. 3B is a transmission electron micrograph of the JB-HEPl cell line at the 90th passage, illustrating glycogen storage by dexamethasone treatment .
  • FIG. 3C is a transmission electron micrograph of the JB-HEPl cell line at the 90th passage, illustrating the bile canaliculi ultrastructure .
  • FIG. 3D is a transmission electron micrograph of the JB-HEPl cell line at the 90th passage, illustrating the D-clathrin lined pits at the basal side of the cells in monolayer culture.
  • FIG. 4 is a Southern Blot analysis of cellular
  • Genomic DNA was extracted from the JB-HEPl cells line and normal fetal liver.
  • lO ⁇ g of each DNA sample were digested with EcoRI, which has a single site in the vector DNA (lanes 1 and 5) .
  • the DNA samples were also digested with Bgll (lanes 2 and 6) , EcoRI and Bgll (lanes 3 and 7) , and PvuII (lanes 4 and 8) . 50, 100, 200, and 500 picograms of plasmid DNA were linearized with EcoRI as a positive control. Hybridization was done with linearized plasmid.
  • FIG. 5 is a Northern Blot analysis of 5 ⁇ g of poly (A) + RNA from the JBH1, JB-HEPl, JBH2 and normal fetal liver, hybridized to a linearized 6.5 kB plasmid.
  • a 2.4 kB normal c-myc transcript is expressed in the immortalized JB-HEPl cell line and the control cells from normal fetal human liver.
  • FIG. 6 shows three autoradiograms of blots illustrating the expression of albumin, ⁇ -fetoprotein and gamm -fibrinogen in the JB-HEPl cell line.
  • this invention relates to the development of an immortal cell line of human hepatic cells derived from normal human fetal liver. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods or materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications and patent documents referenced in this application are incorporated herein by reference . A. PRODUCTION OF CELLS OF THE INVENTION.
  • Human hepatic (liver) tissue useful in the present invention may be obtained as surgical excess tissue from aborted human fetuses at 10 to 24 weeks gestation, preferably at 16 to 20 weeks gestation and most preferably at about 18 weeks gestation. Liver tissue is separated from other fetal tissue by any method known to those skilled in the art, and is preferably taken from the right lobe of the fetal liver.
  • the isolated tissue which will preferably consist mostly of parenchymal hepatic cells, may be minced into smaller pieces and passed through sterile gauze or the like to further separate the cells.
  • the cells may be washed with an antibiotic solution, the selection of which antibiotics will be within the skill of the ordinary artisan, and may be selected from the group consisting of penicillin, kanamycin and amphotericin.
  • the fetal liver cells may be resuspended in a suitable, serum-free medium.
  • the hepatic cells of the present invention are defined as cells that are liver cells or are derived from liver cells, with hepatocytes being preferred.
  • Growth media useful in the present invention is preferably a conditioned growth medium containing hepatocyte growth media (HGM) and MITO+ .
  • MITO+ is commercially available from Collaborative Biomedical Products, in Bedford, Massachusetts, USA.
  • the media may be conditioned by HepG2 cells, and may additionally contain other components, including but not limited to Ham's-F12/M-199 (1:1) supplemented with MITO+, ITS (6 ⁇ g/mL insulin, 6 ⁇ g transferrin and 6 ng/mL selenium) , bovine pituitary extract, insulin and gentamicin sulfate.
  • Cells of the present invention are immortalized by the transfection of an exogenous, cell growth-potentiating gene (i.e., an oncogene) into the cell.
  • an exogenous gene is a nucleic acid sequence not naturally found in the genome of the cell into which the gene is being transfected.
  • the exogenous gene may comprise an entire gene or coding sequence, or some fragment or fragments thereof.
  • the transfected gene is the human c-myc gene.
  • the cells are transfected with a plasmid, and the plasmid is a construct known as pRHPl (see Example 3, below) .
  • the pRHPl construct is a 6.5 kilobase recombinant plasmid containing a portion of the human c- yc gene (exon 2 and exon 3) , is driven by an upstream SV-40 promoter, with both gene fragment and promoter being integrated into a parental pBR322 plasmid.
  • the genes useful in the present invention may be transfected into the hepatic cell by any method known to one skilled in the art, but are preferably transferred into the genome by lipofection.
  • the hepatic cell lines are grown in serum-free or chemically defined medium to minimize the loss of differentiated cell ' functions .
  • the JP-HEP1 cell line comprises cells containing an intact, truncated human c-myc gene driven by an SV40 promoter, as may be confirmed by polymerase chain reaction (PCR) or Southern blot analysis of genomic DNA from the cells. The fidelity of the transfected gene may additionally be confirmed by Northern blot analysis, which indicates the expression of a single, full-length 2.4kB normal c-myc transcript.
  • the JB-HEPl cell line maintains a continuously increased level of c- yc expression (Poly (A)* RNA) of up to about five times the amount expressed by a control specimen (e.g., liver cells of an 18-week, normal human fetus) .
  • c- myc prevents human fetal hepatocytes from entering the terminal differentiated pathway, thus keeping them in a continuous proliferative state.
  • immortalization with the c-myc construct does not prevent the maintenance of a well-differentiated phenotype .
  • the cells of the present invention are of hepatic origin and maintain several critical features of differentiated hepatocytes. Morphological studies reveal the cells to have cytoplasmic organelles and nuclear structure that are more complex than most cultured epithelial cells. Additionally, they possess an ultrastructure similar to the ultrastructure of hepatocytes in vivo . The cells have desmosomes and the cytokeratin complement of secretory epithelial cells. Although the cells express cytokeratins 19 at the 90th passage, they are negative for both keratins 7 and 19 at earlier passages, indicating that they are not like differentiated bile duct epithelial cells.
  • these cells contain glycogen and appear to form structures like bile canaliculi with numerous microvilli at sites of contact between adjacent cells.
  • the cells of the present invention have a subtetraploid karyotype at the 90th passage with relatively few gross chromosomal abnormalities. They express the mRNA for the albumin gene and in early passages express the alpha-fetoprotein gene. When treated with dexamethasone, these cells accumulate large quantities of glycogen, and induce tyrosine amino transferase activity, as has been seen in rodent and human hepatocytes in vi tro . See e . g. , N. Ruiz Bravo et al., Proc . Natl . Acad . Sci . USA 79, 365-368 (1982); M.
  • the immortal hepatic cell line of the present invention is useful as an in vi tro model for replication of human fetal hepatocytes, for the study of genetic regulatory pathways that control hepatocyte differentiation, and for the study of endobiotic and xenobiotic metabolism and the acute phase reaction.
  • the indefinite life span of the human hepatic cell cultures provided by the present invention also makes the cells useful as a more reproducible in vi tro model for the investigation of many facets of human liver function and the development of liver diseases.
  • the cell line may be used as a model for the study of hepatotoxicity and hepatocarcinogenicity, the production of plasma proteins (e.g., albumin) and their expression, the isolation of hepatocyte-specific genes and transcription factors, the effects of hormonal and inflammatory cytokine mediators on the regulation of gene expression, and cytochrome P450 metabolism of therapeutic drugs and hypolipidemic agents. More specific uses of the cells of the instant invention include the following.
  • JB-HEPl cells maintain xenobiotic function associated with "normal hepatocytes
  • cells of the instant invention are uniquely qualified to serve as the basis for in vi tro testing of the response of human hepatocytes to foreign compounds.
  • the intact P450 cytochrome system of JB-HEPl may be employed to study the xenobiotic metabolism of a host of compounds, including drugs, carcinogens, and hormones.
  • the cells of the present invention are responsive to certain mediators of the acute phase reaction in the hu an liver. Accordingly, the cells may be used in developing therapies involved in the treatment of acute phase response (e .g. , drugs, cytokines) .
  • Cells of the invention provide a means to better understand the biology of liver-associated human- restricted pathogens.
  • Cells of the present invention are particularly useful in the investigation of pathogens such as the Hepatitis A, B, C, D, and E viruses, and Plasmodium falciparum, Plasmodium ovale, Plasmodium malariae, and Plasmodium vivax, the latter group of parasites being known to be agents of malaria.
  • the hepatic cells of the present invention may be provided as an in vi tro tissue culture, the tissue culture inoculated with a pathogen such as Plasmodium falciparum or Hepatitis B virus, and the pathogen grown therein. The pathogen may then be collected from the tissue culture and used for the production of antigens or immunogens useful for the production of diagnostic antibodies and for inducing a protective immune response in a suitable subject.
  • Hepatocyte-targeted gene transfer Cells of the invention offer a long-term in vi tro system for developing and studying gene transfer systems targeted to hepatocytes.
  • Liver cell transplantation Cells of the invention are useful for the investigation of long-term storage of isolated hepatocytes by techniques such as cryopreservation. Such stored hepatocytes are useful as repositories of human liver cells, thereby decreasing the dependency on human liver donors for replacement of liver function. Additionally, the cells of the invention may be used to populate a bioartificial liver. Previous attempts to design such bioartificial livers have failed because of the risk of release of cancerous cells into the bloodstream from the tumor-derived cell lines. The cells of the present invention obviate this risk, in that they are not derived from cancer cells but from normal human liver cells.
  • the liver has a major role in lipid metabolism, and thus is involved in the development of atherosclerosis and other cardiovascular disorders. Accordingly, the cells of the present invention may be used for the evaluation of hypolipidemic agents designed to be used in the treatment of cardiovascular disease.
  • the cell line is additionally useful in evaluating drugs that lower LDL cholesterol and increase HDL cholesterol.
  • the cells of the present invention can be used study other aspects of lipid metabolism, for example, to isolate receptors such as the chylomicron remnant receptor, or the genetic regulation of metabolism. Verification of traditional research models.
  • Comparative studies of the human cells of the invention and similar non-human cells are helpful in elucidating interspecies differences which, in turn, are useful for establishing or confirming the value of extrapolating findings obtained from non-human hepatocytes to the analogous human situation.
  • rodent hepatocytes are often used as a model for human hepatocytes
  • research comparing rodent hepatocytes with human cells of the instant invention is particularly valuable.
  • comparative studies of cells of the invention and human hepatoma cell lines such as HepG2 and He3B should also yield important information regarding the reliability of applied research based on in vi tro studies of hepatoma cell lines. As hepatoma cells are transformed cells that do not display the
  • the cell line is additionally useful as a model for the study of graft vs. host disease, which disorder relates to the immunology of transplantation of liver and other organs .
  • mL means milliters
  • L means liters
  • ⁇ L means microliters
  • min means minutes
  • h means hours
  • M means molar
  • mM means millimolar
  • ⁇ M means micromolar
  • mg means milligrams
  • ⁇ g means micrograms
  • mm means millimeters
  • nm means nanometers
  • ⁇ m means micrometers
  • kB means kilobases
  • u means units
  • cpm means counts per minute
  • mL means milliliter
  • kV means kilovolts.
  • Human fetal hepatic cells were obtained from surgical excess liver tissue from a fetus at 18 weeks gestation, according to the institutional guidelines for the protection of human subjects at the University of North Carolina-Chapel Hill. A small piece of the liver was separated from the connective tissue and the gallbladder. Then the remainder of the tissue, was minced into small pieces with scalpel blades. The minced tissue was passed through folded sterile gauze, and the cells and tissue fragments were washed three times by centrifugation 75 X g for 10 min, in OptiMem (Gibco-BRL Laboratories, Grand Island, New York, USA) containing an antibiotic mixture of 100 ⁇ g/mL penicillin G, kanamycin and amphotericin B.
  • OptiMem Gibco-BRL Laboratories, Grand Island, New York, USA
  • the preparation used for culture was composed of single cells and cell aggregates.
  • the mixture of single cells and cell aggregates (totaling approximately 2 grams of tissue) was resuspended in 50mL of HGM (hepatocyte growth medium) and 4% FBS (Fetal Bovine Serum) .
  • 5mL of cell suspension was plated in each of ten, 60mm culture dishes (Corning) and incubated for 24 hours in a humidified atmosphere of 5% C0 2 in air at 37°C.
  • HBSS Hanks' Balanced Salt Solution
  • CON/HGM 20% conditioned serum-free medium
  • MIT0+ per manufacturer's recommendation
  • ITS 6 ⁇ g/mL insulin, 6 ⁇ g transferrin and 6ng/mL selenium
  • bovine pituitary extract 5 ⁇ g/mL, all from Collaborative
  • the plasmid pRHPl which was rationally provided by Dr. Russel Kaufman (Duke University) , is a 6.5 kB construct containing a portion of human c-myc gene (exon 2 and exon 3) driven by an upstream SV-40 promoter cloned into the pBR322 plasmid.
  • the human fetal hepatic cells were plated in ten 60mm culture dishes, and transfection was accomplished by lipofection of the primary cultures on the third day after plating.
  • Cell-s were transfected with a mixture of 15 ⁇ l of Lipofectin® (Gibco-BRL, Grand Island, New York, USA) and buffer containing 10 ⁇ g of plasmid DNA that had been linearized with EcoRI.
  • the DNA and Lipofectin® were mixed according to the manufacturer's instructions, then resuspended in 3 mL of serum-free OptiMem per 60 mm dish. After a 15 min incubation the mixture of linearized plasmid DNA and Lipofectin® was added to six 60mm dishes. Four control dishes were treated in the same manner, except that linearized plasmid DNA was not added to the Lipofectin and medium mixture.
  • the ten dishes were incubated for a period of 24 h under a humidified atmosphere of 5% C0 2 in air at 37°C. After 24 h, medium was replaced with 5mL of 20% conditioned HGM per dish. The culture medium was replaced three times per week.
  • Transfection of the primary cultures of human fetal hepatic cells was performed three days after attachment. Proliferating cells spread out on the plastic culture plates and differences between the transfected dishes and nontransfected controls could be detected only after about four weeks. Cells in the nontransfected cultures become vacuolated, ceased to replicate, and detached from the culture plates. In contrast, the transfected cells remained attached to the plates and were polyhedral in shape, often in a cordlike appearance reminiscent of hepatic trabeculae with a centrally located spherical nucleus. See FIG. 1A. Many cells were binucleate. Nuclei contained one or more nucleoli and scattered heterochromatin clumps. A rim of perinuclear heterochromatin was also prominent, as seen in FIGS. IB and IC.
  • Colonies of tightly packed cells which proliferated and reached a diameter of 1 cm or more within 6-8 weeks were observed in once dish containing the c-myc transfected cells. Similar cells were not found in the control dishes. Cells from a plate with established colonies were released from the plate by treatment with a 1:1 mixture of collagenase Type A at 250 ⁇ g/mL (Boehringer-Mannheim, Indianapolis, Indiana, USA) and 5000 u/mL of dispase (Collaborative Research) .
  • the cell suspension was centrifuged at 75 x g for 5 min, and the pellet was resuspended in unsupplemented growth medium (1:1 mixture of Ham's F12 and M199 and 4% Bovine Seru ) to wash away the collagenase and dispase mixture. The washing procedure was repeated three times.
  • the cells were cultured and expanded in 20% conditioned hepatocyte growth medium (CON/HGM) . The ability of the transfected cells to form colonies on a background of senescing normal cells was the only selective procedure used.
  • the expanded cell culture was designated the JBHl parental cell line.
  • 10 dishes of the parental cell line JBHl had 25-50 single cells per dish remaining attached to the plastic. Rather than discarding these dishes as usual, the dishes were fed normally and the single cells were marked and their growth into colonies was followed. After 6 weeks, 33 colonies were isolated individually because of their hepatocyte-like morphology, based on their apparent trabecular pattern of cell growth.
  • One clone, designated JB-HEPl was kept in culture, while the rest were cryopreserved.
  • the clonal cell line JB- HEPl has been propagated for six years, over 250 passages (more than 500 doublings) .
  • the karyotype of the JB-HEPl human immortalized hepatic cell line was determined based on the examination of over fifty metaphase spreads that were banded by the trypsin-Giemsa method. Briefly, exponentially growing cells at about 70% confluence were treated with colcemid (0.02 ⁇ g/mL, Gibco-BRL) for an hour, at 37°C then removed from culture dishes by trypsinization. The cells were suspended in hypotonic (0.075 M) KC1 for 20 min at 37°C and fixed 3 times with Carnoy's fixative (3:1 methanol/acetic acid). Fixed cells were dropped onto glass slides to create metaphase spreads.
  • the slides were treated with trypsin (lmg/mL in isotonic buffered saline) for one minute at room temperature and banded with Giemsa stain for five min at room temperature .
  • Metaphase chromosomes were photographed at 10OX using 400 speed Kodak t-max film.
  • the JB-HEPl hepatocyte cell line showed a prevalence of near tetraploid cells containing a modal number of 69 chromosomes.
  • a typical karyotype for the cells at passage 90 is shown in FIG. 2. Although most of the chromosomes appeared to have no detectable abnormalities, some spreads contained marker chromosomes with structural translocations that were not identified for chromosomal origin.
  • Example 6 Immunohistochemistry The cells were released by trypsinization and washed in HBSS. 1 x 10 4 cells were cytospun onto glass slides (Fisher, Pittsburgh, Pennsylvania, USA) . Antibodies to cytokeratins 8 and 18, and CAM 5.2 (Becton Dickinson, San Jose, California, USA) were used at a 1:10 dilution. Antibodies to cytokeratins 7 and 19
  • Mature hepatocytes normally express the neutral cytokeratin 8 and the acidic cytokeratin 18. During early fetal development hepatocytes also express keratin 19. See P. Stosiek et al . , Liver 10, 59-63 (1990) . After the 10th week of gestation, keratin 19 is found only in the bile duct cells and it continues to be expressed in bile duct cells in the adults. The fact that these cells ceased to express keratin 19 at an early stage in culture then reexpressed it after further passage may reflect early maturation of hepatocytes in vitro followed later by the loss of this differentiated feature with extensive maintenance in culture . Similarly, these cultured cells expressed the message for alpha-feto protein early during their progagation in vitro but ceased to express it when evaluated at the
  • cells were plated on plastic culture dishes (Lux-Nunc, Naperville, Illinois, USA) at low density and allowed to proliferate until they reached confluence.
  • the cultured cells were fixed in situ in 3% gluteraldehyde in serum-free HGM pH 7.2 at 37°C for one hour and post-fixed in 1% osmium tetroxide in 0.1M sodium phosphate buffer, pH7.4 or 1% osmium tetroxide/1.25% potassium ferrocyanide in 0.1M sodium phosphate buffer pH 7.4.
  • Fixed cells were dehydrated through a graded series of ethanol washes, and then embedded in PolyBed 812 (Polysciences Inc., Warrington, Pennsylvania, USA) .
  • the plastic blocks were separated from the culture plates, 70nm ultrathin section were cut, mounted on uncoated copper grids, and double stained with uranyl acetate and lead citrate. The sections were examined at 70Kv with Zeiss Em 10 electron microscope (Carl Zeiss Inc., Oberkochen, Germany) at the magnification indicated.
  • the tubular elements of this organelle contain small, dense spherical particles like newly synthesized very low density serum lipoprotein.
  • the cells had a centrally placed ovoid nucleus with one or several nucleoli.
  • the nuclear structure contained a dense network of granular strands of heterochromatin at the periphery and a double nuclear membrane with distinct nuclear pores. Adjacent cells were joined by desmosomes . Numerous intercellular spaces lined with microvilli resembling bile canaliculi, (FIG. 3C) could also been seen between cells this suggest that the hepatocytes are polarized in culture.
  • Glycogen granules were present and were arranged in clusters to form "rosettes". See FIG. 3B.
  • the ultrastructure of the JB-HEPl cell line demonstrates a phenotype with several features of differentiated human hepatocytes .
  • Example 8 DNA analysis High molecular weight DNA was prepared from tissue samples and confluent cell cultures by standard procedures. 10 ⁇ g of genomic DNA digested with EcoRI, was fractionated on a 0.8% agarose gel and transferred to a nylon membrane (Hybond-N, Amersham Life Sciences) for hybridization. The membranes were hybridized with a radiolabelled probe prepared with the linearized plasmid used in the transfection, at a specific radioactivity of 10 6 cpm/mL in 3x SSC at 42°C overnight (lxSSC, 0.15M NaCl , 0.015M trisodium citrate) . The membranes were washed at (high stringency 0.5x SSC at 55°C) , dried and autoradiographed for 1-3 days at-70°C using Kodak XOMAT XAR-5 film.
  • the JB-HEPl cell line was evaluated to distinguish it from several other human specimens and other human cells in culture in the laboratory by evaluating one of their variable genetic markers.
  • the human origin of the JB-HEPl cell line was verified based on the presence of sequences detected by the variable tandem repeat (VNTR) probe pYNH24 (American Type Culture Collection, Rockville, Maryland, USA) on a Southern blot of EcoRI-restricted DNA from the JB-HEPl cell line.
  • VNTR variable tandem repeat
  • the cells have a different VNTR pattern than the HepG2 cell line used to condition medium, and thus they are not a contaminant derived from these cells.
  • Southern blot analysis of c-myc of the DNA of the JB-HEPl cells was done using the EcoRI linearized plasmid to detect the transfected copies of c-myc. The comparative magnitudes of the label hybridized in the Southern blots was quantitated by densitometry of the autoradiograms . These results indicate that at least a single complete linearized copy of the exogenous c-myc gene is integrated in the genome of the JB-HEPl cell line. See FIG. 4.
  • the integration of the c-myc fragment into the JB-HEPl was verified by PCR analysis, using a primer homologous to a segment of the upstream SV40 promoter and a primer to the complementary DNA strand homologous to a sequence in the second exon of c-myc .
  • the results show that an amplified product of the same size as that produced with the plasmid as template could be generated from JB-HEPl cells, whereas it is absent from the untransfected control cells.
  • Internal PCR controls verified that PCR amplifications could achieved from the control DNA samples .
  • RNA was prepared from CsCl-purified total RNA using oliog-dT cellulose chromatography kits (Pharmacia LKB Biotechnology, Piscataway, New Jersey, USA) according to the manufacturer's instructions.
  • oliog-dT cellulose chromatography kits Pharmacia LKB Biotechnology, Piscataway, New Jersey, USA
  • Poly (A) + for the c-myc expression (20 ⁇ g/lane for the former and 5 ⁇ g/lane for the latter) were fractionated by electrophoresis in 1.0 M formaldehyde, 1% agarose gels for transfer to nylon membranes . After the gel was washed for 1 h in 3 gel volumes of 2Ox SSC with one wash change after 30 minutes, the RNA was transferred to nylon membrane (Hybond-N®, Amersham Life Sciences) by capillary blotting for 24 h in 20xSSC. RNA was cross- linked to air dried nylon membranes by UV exposure for one min on a UV-Stratalinker-1800 (STRATAGENE).
  • Poly (A) + selection of total RNA from confluent immortalized cultured hepatocytes was required in order to detect and quantify transcripts of the albumin, fibrinogen and alpha-fetoprotein genes.
  • the positive control for each Northern blot was 20 ⁇ g of total mRNA or a 5ug aliquot of poly (A) + RNA isolated from fresh human fetal liver (18 weeks gestation) . After suitable autoradiographic exposure, the membranes were stripped of the probe by washing three times in 0.1% SDS at 100°C, and re- hybridized with a new probe as before. See FIG. 5.
  • the JB-HEPl cell line at passage 90 was evaluated for the expression of transcripts for the albumin and alpha-fetoprotein genes as well as gamma- fibrinogen (FIG. 6) .
  • Albumin was expressed strongly in the JB-HEPl cells like in the normal fetal hepatocytes and HEPG2 cells.
  • the c-myc was expressed at a high level, 5-fold over control hepatocytes from a normal 18 week fetus.
  • the expression of the alpha- fetoprotein gene could not be detected at passage 90, whereas this gene had been expressed in the parental cells at passage 30.
  • the JB-HEPl cell line expressed the following: albumin (2.2KB +) , gamma-fibrinogen (2.2KB +) , and alpha-fetoprotein.
  • Tyrosine aminotransferase (TAT) activity was evaluated because it is a typical example of hormonal regulation of gene function in the liver, and is an indicator of the involvement and integrity of the glucocorticoid receptor in these cells.
  • TAT enzyme activity were evaluated in the JB-HEPl cells at the 90th passage both with and without prior induction with 10 "7 M dexamethasone for 24 hours in culture.
  • Treatment of the JB-HEPl cell line for a period of 24 h with 10 "7 M dexamethasone induced the TAT enzyme activity by 11.8-fold as compared to the basal level in uninduced JB-HEPl cells.
  • Tyrosine amino transferase activity is restricted to hepatocytes but is normally undetectable before birth. See 0. Greengard, Biochemical Actions of Hormones Vol . 1 , 53-87 (1970). The cultured cells analyzed above had a low but detectable level of TAT activity. One interpretation of these results is that these cells have undergone post-fetal maturation in vi tro . The results further suggest that these cells, when treated with dexamethasone, can induce TAT activity and accumulate glycogen, indicating that they remain responsive to some of the gene regulatory processes that operate in normal human hepatocytes.

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Abstract

L'invention concerne une lignée cellulaire hépatique humaine immortalisée. On crée cette lignée cellulaire par lipofection de cultures primaires de cellules hépatiques foetales humaines, à l'aide d'un plasmide contenant le second et le troisième exon du gène humain c-myc entraîné par un promoteur du SV-40. Des analyses cytochimiques, morphologiques et phénotypiques ont montré que la lignée cellulaire résultante possédait des caractéristiques hépatocytaires. Lorsque l'on a analysé ces cellules après 90 passages, elles ont continué d'exprimer des marqueurs hépatocytaires choisis, notamment l'albumine et le gamma-fibrinogène. La microscopie électronique par transmission révèle que les cellules possèdent des caractéristiques épithéliales, notamment des jonctions desmosomales et de nombreuses microvillosités de surface, et qu'elles forment des structures qui ressemblent à des canalicules biliaires. L'invention concerne également des procédés de préparation de cette lignée cellulaire hépatique immortalisée.
PCT/US1997/019764 1996-08-30 1997-08-21 Lignee cellulaire hepatique humaine immortalisee et procede de preparation associe Ceased WO1998058050A1 (fr)

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AU50054/97A AU5005497A (en) 1996-08-30 1997-10-21 Immortalized human hepatic cell line and method of making the same

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US2512096P 1996-08-30 1996-08-30
US60/025,120 1996-08-30

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WO1998058050A1 true WO1998058050A1 (fr) 1998-12-23

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PCT/US1997/019764 Ceased WO1998058050A1 (fr) 1996-08-30 1997-08-21 Lignee cellulaire hepatique humaine immortalisee et procede de preparation associe
PCT/US1997/014745 Ceased WO1998008935A1 (fr) 1996-08-30 1997-08-21 Lignee cellulaire hepatique humaine immortelle

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CN107043738A (zh) * 2017-02-07 2017-08-15 韶关学院 一种猪肝细胞无血清培养基及其制备方法

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Publication number Priority date Publication date Assignee Title
US6653105B2 (en) 1998-02-11 2003-11-25 Vitagen, Inc. Clonal cells and cell lines derived from C3A cells and methods of making and using them
CA2329857A1 (fr) 1998-04-28 1999-11-04 Takeda Chemical Industries, Ltd. Lignee cellulaire hepatique immortalisee derive d'origine humaine
GB0420963D0 (en) * 2004-09-21 2004-10-20 Reneuron Ltd Hepatocyte
WO2007035082A1 (fr) * 2005-09-23 2007-03-29 Hep-Art Medical Devices B.V. Lignées cellulaires de foie fœtal immortalisées
CN102643777A (zh) 2006-01-04 2012-08-22 巴克斯特国际公司 无寡肽的细胞培养基
CN103087989A (zh) * 2013-02-01 2013-05-08 湖南省肿瘤医院 一种人肝癌细胞系hlcz01及其应用

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DE69106353T2 (de) * 1990-04-03 1995-07-13 Southwest Found Biomed Res Immortalisierte primate hepatozyte zellinie.

Non-Patent Citations (2)

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Title
ADAMS et al., "The C-myc Oncogene Driven by Immunoglobulin Enhancers Induces Lymphoid Malignancy in Transgenic Mice", NATURE, 12 December 1988, Vol. 318, pages 533-538. *
KINSELLA et al., "Introduction of the Activated N-Ras Oncogene Into Human Fibroblasts by Retroviral Vector Induces Morphological Transformation and Tumorigenicity", CARCINOGENESIS, 1990, Vol. 11, No. 10, pages 1803-1809. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107043738A (zh) * 2017-02-07 2017-08-15 韶关学院 一种猪肝细胞无血清培养基及其制备方法

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AU5005497A (en) 1999-01-04
AU4081997A (en) 1998-03-19

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