Hepatocyte precursor cell lines
First Claim
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1. A method of preparing hepatic cells that express mature hepatic cell markers comprising:
- a) culturing a population of cells from liver tissue obtained from a mammal, wherein the population of cells comprises stellate cells, hepatocytes, cholangiocytes, oval cells, Kupffer cells, sinusoidal endothelial cells and hepatic precursor cells,b) enriching the population of cells for hepatic precursor cells,c) culturing the enriched population of cells in a first culture medium comprising a guanine nucleotide precursor of at least 50 μ
M, or an analogue or derivative thereof, wherein said guanine nucleotide precursor suppresses asymmetric cell kinetics thereby allowing exponential growth of said hepatic precursor cells;
d) passaging said cultured hepatic precursors in the culture medium of step (c) to allow expansion of said hepatic precursor cells; and
e) contacting the expanded hepatic precursors of step (c) with a second culture medium that is free from the guanine nucleotide precursor of the first medium, and selecting cells with expression of a mature hepatic cell marker, wherein the hepatic cell marker is selected from the group consisting of;
H4 antigen, α
1-antitrypsin (AAT), cytokeratin 7 (CK7), cytokeratin 8 (CK8), albumin cytochrome p450 1A1, hepatocyte nuclear transcription factor (HNF-3), CAAT enhancer-binding protein (CEBP)-alpha and CAAT enhancer-binding protein (CEBP)-beta.
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Abstract
The present invention is directed to methods for readily generating hepatocyte precursor cell lines that retain hepatocyte-specific functions after extensive in vitro culturing. The methods comprise isolating and culturing hepatocyte precursor cell lines under permissive culture conditions that suppress asymmetric cell kinetics and allow exponential growth of the precursor cells, followed by transferring the hepatocyte precursor cell lines to non-permissive culture conditions that allow expression of asymmetric cell kinetics and induce expression of hepatocyte-specific characteristics.
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6 Claims
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1. A method of preparing hepatic cells that express mature hepatic cell markers comprising:
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a) culturing a population of cells from liver tissue obtained from a mammal, wherein the population of cells comprises stellate cells, hepatocytes, cholangiocytes, oval cells, Kupffer cells, sinusoidal endothelial cells and hepatic precursor cells, b) enriching the population of cells for hepatic precursor cells, c) culturing the enriched population of cells in a first culture medium comprising a guanine nucleotide precursor of at least 50 μ
M, or an analogue or derivative thereof, wherein said guanine nucleotide precursor suppresses asymmetric cell kinetics thereby allowing exponential growth of said hepatic precursor cells;d) passaging said cultured hepatic precursors in the culture medium of step (c) to allow expansion of said hepatic precursor cells; and e) contacting the expanded hepatic precursors of step (c) with a second culture medium that is free from the guanine nucleotide precursor of the first medium, and selecting cells with expression of a mature hepatic cell marker, wherein the hepatic cell marker is selected from the group consisting of;
H4 antigen, α
1-antitrypsin (AAT), cytokeratin 7 (CK7), cytokeratin 8 (CK8), albumin cytochrome p450 1A1, hepatocyte nuclear transcription factor (HNF-3), CAAT enhancer-binding protein (CEBP)-alpha and CAAT enhancer-binding protein (CEBP)-beta. - View Dependent Claims (2, 3, 4, 5, 6)
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Specification