Differentiated human liver cell cultures and their use in bioartificial liver systems

Abstract

Human hepatocyte cell cultures and their use in bioreactors and bioartificial liver (BAL) systems are provided. The cells have constitutive liver-specific metabolic activity resembling that of freshly isolated human hepatocytes. The hepatocyte cells and BAL systems can be used to treat patients suffering from acute liver failure, end-stage liver disease, or acute-on-chronic liver disease.

1 . A cell culture comprising differentiated cells from a human hepatocyte cell line in a suitable culture medium, wherein said differentiated cells have constitutive liver-specific metabolic activity, the cell culture being obtained by a process comprising:
 selecting a human hepatocyte cell line;   a phase of cell proliferation comprising culturing cells of said human hepatocyte cell line in a suitable culture medium, preferably comprising serum, hormones, growth-factors and antibiotics;   loading viable cells to an amount of at least 5% of normal liver mass in a bioreactor comprising a three dimensional support matrix, and allowing the cells to attach to said matrix;   an expansion or proliferation phase comprising culturing the cells loaded in the bioreactor in a suitable culture medium, preferably comprising serum, hormones, growth-factors and antibiotics, to achieve at least one cell population doubling; and   a phase of cell differentiation comprising culturing the cells in a culture medium comprising serum, hormones, growth factors and antibiotics.   
     
     
         2 . The cell culture according to  claim 1 , wherein the cell differentiation phase lasts at least 7 days. 
     
     
         3 . The cell culture according to  claim 1 , wherein the three dimensional support matrix comprises a porous sheet or mat, a fiber network matrix, an open-pore foam, or a semi-solid material. 
     
     
         4 . The cell culture according to  claim 1 , wherein said human hepatocyte cell line is HepaRG, deposited at the Collection Nationale de Cultures de Microorganismes, Institut Pasteur, Accession No. 1-2652. 
     
     
         5 . The cell culture according to  claim 1 , wherein the phase of cell differentiation comprises culturing the cells in a suitable culture medium comprising at least 0.5 mM carbamoyl glutamate. 
     
     
         6 . The cell culture according to  claim 1 , wherein the phase of cell differentiation comprises culturing the cells in a suitable culture medium that is substantially free of DMSO. 
     
     
         7 . A bioreactor, comprising a chamber or vessel holding a matrix loaded with the cell culture of  claim 1 , the bioreactor further comprising means for oxygenating the matrix. 
     
     
         8 . A bioartificial liver device, comprising the bioreactor of  claim 7 , a liquid circuit, a plasma separator or plasmapheresis unit, and one or more pumps. 
     
     
         9 . A method of producing the bioreactor of  claim 7 , comprising the steps of:
 a phase of cell proliferation comprising culturing cells of a human hepatocyte cell line in a suitable culture medium, preferably comprising serum, hormones, growth-factors and antibiotics;   loading viable cells to an amount of at least 5% of normal liver mass in a bioreactor comprising a three dimensional support matrix, and allowing the cells to attach to said matrix;   an expansion or proliferation phase comprising culturing the cells in a suitable culture medium, preferably comprising serum, hormones, growth-factors and antibiotics, to achieve at least one cell population doubling; and   a phase of cell differentiation comprising culturing the cells in a culture medium comprising serum, hormones, growth factors and antibiotics.   
     
     
         10 . The method according to  claim 9 , wherein said human hepatocyte cell line is HepaRG, deposited at the Collection Nationale de Cultures de Microorganismes, Institut Pasteur, Accession No. 1-2652. 
     
     
         11 . The method according to  claim 9 , wherein the three dimensional support matrix comprises a porous sheet or mat, a fiber network matrix, an open-pore foam, or a semi-solid material. 
     
     
         12 . The method according to  claim 9 , wherein the culture medium used in the cell differentiation phase comprises at least 0.5 mM carbamoyl glutamate. 
     
     
         13 . The method according to  claim 9 , wherein the cell differentiation phase lasts at least 7 days. 
     
     
         14 . The method according to  claim 9 , wherein the culture medium is substantially free of DMSO. 
     
     
         15 . A method of treating a subject suffering from acute liver failure, end-stage liver disease, or acute-on-chronic liver disease, the method comprising extra-corporeal liver support by perfusing the subject's plasma through the bioreactor of  claim 12 . 
     
     
         16 . The method according to  claim 15 , wherein the method does not comprise any steps in which carbamoyl glutamate is administered to the bioreactor and/or to the subject's plasma. 
     
     
         17 . A process of producing a cell culture comprising:
 selecting a human hepatocyte cell line;   a phase of cell proliferation comprising culturing cells of said human hepatocyte cell line in a suitable culture medium, preferably comprising serum, hormones, growth-factors and antibiotics;   loading viable cells to an amount of at least 5% of normal liver mass in a bioreactor comprising a three dimensional support matrix, and allowing the cells to attach to said matrix;   an expansion or proliferation phase comprising culturing the cells loaded in the bioreactor in a suitable culture medium, preferably comprising serum, hormones, growth-factors and antibiotics, to achieve at least one cell population doubling; and   a phase of cell differentiation comprising culturing the cells in a culture medium comprising serum, hormones, growth factors and antibiotics.   
     
     
         18 . The process according to  claim 17 , wherein said human hepatocyte cell line is HepaRG, deposited at the Collection Nationale de Cultures de Microorganismes, Institut Pasteur, Accession No. 1-2652. 
     
     
         19 . The process according to  claim 17 , wherein the cell differentiation phase lasts at least 7 days. 
     
     
         20 . The process according to  claim 17 , wherein the culture medium is substantially free of DMSO. 
     
     
         21 . The process according to  claim 17 , wherein the three dimensional support matrix comprises a porous sheet or mat, a fiber network matrix, an open-pore foam, or a semi-solid material. 
     
     
         22 . The process according to  claim 17 , wherein the phase of cell differentiation comprises culturing the cells in a suitable culture medium comprising at least 0.5 mM carbamoyl glutamate.