Long-term three dimensional tissue culture system

• US 6,737,270 B1
• Filed: 12/07/1999
• Issued: 05/18/2004
• Est. Priority Date: 12/07/1999
• Status: Expired due to Fees

• Current Assignee: University Of Pittsburgh (University of Pittsburgh of The Commonwealth System of Higher Education)
• Original Assignee: University of Pittsburgh of The Commonwealth System of Higher Education
• Inventors: Michalopoulos, George, Bowen, William C.
• Primary Examiner(s): Naff, David M.
• Application Number:
  US09/455,952
• Time in Patent Office: 1,624 Days
• Field of Search: 435/177, 435/180, 435/395, 435/373, 435/402, 435/405, 424/93.7
• US Class Current: 435/373
• CPC Class Codes: A01N 1/0231 Chemically defined matrices, e.g. alginate gels, for immobilising, holding or storing cells, tissue or organs for preservation purposes; Chemically altering or fixing cells, tissue or organs, e.g. by cross-linking, for preservation purposes
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61L 27/3804 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
• CPC Class Codes: A61L 27/3839 characterised by the site of application in the body materials for artificial blood vessels A61L27/507; materials for use in artificial skin A61L27/60
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/12 Hepatocyte growth factor [HGF]
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2502/14 hepatocytes
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0671 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells

Abstract

A method is provided for long term culture of proliferating hepatocytes that retain hepatic function to produce a hepatic cell culture. Hepatocytes and nonparenchymal cells are co-cultured ex vivo on a matrix coated with a molecule that promotes cell adhesion, proliferation or survival, in the presence of growth factors, resulting in a long-term culture of proliferating hepatocytes that retain hepatic function. The co-culturing method results in the formation of matrix/hepatic cell clusters that may be mixed with a second structured or scaffold matrix that provides a three-dimensional structural support to form structures analogous to liver tissue counterparts. The method can be used to form bio-articial livers through which a subjects blood is perfused. In an embodiment, the hepatocytes and nonparenchymal cells are derived from disaggregated liver tissue and are co-cultured in the presence of epidermal growth factor or heptocyte growth factor and beads coated with extracellular matrix protein, Alternatively, the hepatic cell culture may be implanted into the body of a recipient host having a hepatic disorder. Such hepatic disorders, include, for example, cirrhosis of the liver, induced hepatitis, chronic hepatitis, primary sclerosing cholangitis and alpha₁ antitrypsin deficiency.

Claims

1. A population of hepatocytes and nonparenchymal cells, derived using a method comprising:

co-culturing hepatocytes and nonparenchymal cells, derived from disaggregated liver tissue, in the presence of

(a) one or more growth factors that support the growth of hepatocytes comprising epidermal growth factor or hepatocyte growth factor and

(b) beads coated with extracellular matrix protein that promotes cell adhesion under conditions sufficient to allow for the proliferation of said hepatocytes while retaining hepatic faction of said hepatocytes.

2. A method for generating a hepatic cell culture comprising:

co-culturing hepatocytes and nonparenchymal cells derived from disaggregated liver tissue, in the presence of

(a) one or more growth factors that support the growth of hepatocytes comprising epidermal growth factor or hepatocyte growth factor and

(b) beads coated with extracellular matrix protein that promotes cell adhesion under conditions sufficient to allow for the proliferation of said hepatocytes while retaining hepatic function of said hepatocytes.

3. The method of claim 2 wherein the beads are polystyrene beads.

4. The method of claim 2 wherein the extracellular matrix protein is type I collagen.

5. The method of claim 2 wherein the growth factor is epidermal growth factor.

6. The method of claim 2 wherein the growth factor is hepatocyte growth factor.