Methods for Producing Cells Having a Phenotype of a Primary Human Hepatocytes and Compositions
AssigneeThe Governors of the University of Alberta
InventorsD. Lorne Tyrrell, Hendrikje Geesje Steenbergen, Michael A. Joyce
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Abstract
The present disclosure provides methods and compositions relating to in vitro cultures of human hepatocyte cell lines which exhibit a primary human hepatocyte phenotype. Such cell lines are susceptible to infection by a hepatotrophic virus, such as HCV or HBV, and support both viral replication and high levels of viral particle production. Such in vitro cultures find use in production and study of hepatotrophic virus, as well as methods of screening (e.g., for antiviral drugs, assessing drug metabolism), and study of primary human hepatocytes.
Claims40
A method of producing a cell culture comprising cells having a primary human hepatocyte phenotype, the method comprising:
- culturing a human hepatocellular carcinoma (hHCC) cell line in a culture medium comprising human serum for more than 11 days
- wherein said culturing induces differentiation of the hHCC cell line into a cell having a primary human hepatocyte phenotype.
A cell culture comprising:
- cells having a phenotype of a human primary hepatocyte, wherein the cells are the differentiated progeny of an hHCC cell line;
- and
- a culture medium comprising human serum.
A method of producing viral particles, the method comprising:
- incubating a cell culture comprising a human hepatocellular carcinoma (hHCC) cell line in a culture medium comprising human serum for more than 11 days.
- wherein said incubating induces differentiation of the hHCC cell line into a cell having a primary human hepatocyte phenotype;
- introducing a genome of a hepatotrophic virus into at least one of the hHCC cell line or the cell having primary human hepatocyte phenotype;
- and
- maintaining the cell culture under conditions suitable for production of viral particles.
A virally-infected cell culture comprising cells produced by a method comprising:
- incubating a cell culture comprising a human hepatocellular carcinoma (hHCC) cell line in a culture medium comprising human serum for more than 11 days, wherein said culturing induces differentiation of the hHCC cell line into a cell having a primary human hepatocyte phenotype;
- and
- introducing a genome of a hepatotrophic virus into at least one of the hHCC cell line or the cell having primary human hepatocyte phenotype.
A method for screening a candidate agent for antiviral activity, the method comprising:
- incubating a cell culture comprising a human hepatocellular carcinoma (hHCC) cell line in a culture medium comprising human serum for more than 11 days, wherein said incubating induces differentiation of the hHCC cell line into a cell having a primary human hepatocyte phenotype;
- introducing a genome of a hepatotrophic virus into at least one of the hHCC cell line or the cell having primary human hepatocyte phenotype;
- contacting the cell culture with a candidate antiviral agent;
- maintaining the cell culture under conditions suitable for viral replication;
- and
- detecting the presence or absence of an effect of the candidate agent upon viral replication;
- wherein a decrease in viral particle production in the presence of the candidate agent as compared to the absence of the candidate agent indicates the candidate agent have antiviral activity.
A method for screening a sample suspected of containing an antibody for antiviral activity, the method comprising:
- incubating a cell culture comprising a human hepatocellular carcinoma (hHCC) cell line in a culture medium comprising human serum for more than 11 days, wherein said incubating induces differentiation of the hHCC cell line into a cell having a primary human hepatocyte phenotype;
- introducing a genome of a hepatotrophic virus into at least one of the hHCC cell line or the cell having primary human hepatocyte phenotype;
- contacting the cell culture with a sample of suspected of containing an antibody;
- maintaining the cell culture under conditions suitable for viral replication;
- and
- detecting the presence or absence of an effect of the sample upon viral replication;
- wherein a decrease in viral particle production in the presence of the sample as compared to the absence of the sample indicates the sample contains an antibody having antiviral activity.
A method for screening for a candidate agent for the treatment of a lipoprotein mediated disease, the method comprising:
- incubating a cell culture comprising a human hepatocellular carcinoma (hHCC) cell line in a culture medium comprising human serum for at least 3 days, wherein said incubating induces differentiation of the hHCC cell line into a cell having a primary human hepatocyte phenotype;
- contacting the cell culture with the candidate agent;
- and
- assaying the cell culture for the presence or absence of an effect of the candidate agent on the levels of lipoprotein secreted by the differentiated hHCC cell line as compared to a control sample;
- wherein an effect of the candidate agent on the levels of lipoprotein secreted by the differentiated hHCC cell line indicates that the candidate agent can be used for the treatment of the lipoprotein mediated disease.
A method of infecting a cultured hepatocytic cell with a hepatotrophic microorganism, the method comprising:
- contacting a cultured hepatocyte cell with an infectious hepatotrophic microorganism in a culture medium comprising serum depleted of LDL-receptor binding lipoproteins;
- wherein said contacting is for a time sufficient to provide for infection of the cultured hepatocyte cell with the hepatotrophic microorganism.
Dependencies are inferred from the claim text.