Novel long-term three-dimensional tissue culture system

• US 20030096411A1
• Filed: 10/28/2002
• Published: 05/22/2003
• Est. Priority Date: 12/07/1999
• Status: Abandoned Application

• Current Assignee: University Of Pittsburgh (University of Pittsburgh of The Commonwealth System of Higher Education)
• Original Assignee: University Of Pittsburgh (University of Pittsburgh of The Commonwealth System of Higher Education)
• Inventors: Michalopoulos, George, Bowen, William C. Jr.
• Application Number:
  US10/281,575
• Publication Number:
  US 20030096411A1
• Time in Patent Office: Days
• 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 2502/253 Bladder cells
• CPC Class Codes: C12N 5/0671 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells

Abstract

The present invention relates to a novel tissue culture system that provides for the long term culture of proliferating hepatocytes that retain hepatic function. Disclosed are methods and compositions for ex vivo culturing of hepatocytes and nonparenchymal cells 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 hepatic cell culture system can be used to form bio-artificial livers through which a subjects blood is perfused. Alternatively, the novel hepatic cell culture system 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 method for generating a hepatic cell culture comprising co-culturing hepatocytes and nonparenchymal cells, in the presence of growth factors corticosteroid and a matrix coated with at least one biologically active molecule that promotes cell adhesion, proliferation or survival under conditions sufficient to allow for the proliferation of hepatocytes that retain hepatic function.

2. The method of claim 1 wherein the hepatocytes and nonparenchymal cells are derived from a liver tissue sample.

3. The method of claim 1 wherein the matrix is in the form of polystyrene beads.

4. The method of claim 1 wherein the matrix is coated with an extracelluar matrix protein.

5. The method of claim 1 wherein the matrix is coated with type I collagen.

6. The method of claim 1 wherein the growth factor is epidermal growth factor.

7. The method of claim 1 wherein the corticosteroid is dexamethasone.

8. The method of claim 1 wherein the growth factor is hepatocyte growth factor.

10. The method of claim 8 wherein the hepatocytes and nonparenchymal cells derived from a liver tissue sample.

11. The method of claim 8 wherein the matrix is in the form of a biomatrix gel.

12. The method of claim 8 wherein the matrix is coated with an extracelluar matrix protein.

13. The method of claim 1 wherein the matrix is coated with type I collagen.

14. The method of claim 8 wherein the matrix further comprises growth factors incorporated into said matrix.

9. A method for generating a three-dimensional hepatic cell culture system comprising:

contacting a three-dimensional support matrix with a hepatic cell culture comprising hepatocytes and nonparenchymal cells bound to a matrix coated with at least one biologically active molecule that promotes cell adhesion, proliferation or survival;

under conditions sufficient to allow for the proliferation of the hepatic cell culture to form a three-dimensional hepatic cell structure.

15. A population of matrix/hepatic cell clusters comprising hepatocytes and nonparenchymal cells associated with a matrix coated with at least one biologically active molecule that promotes cell adhesion, proliferation or survival.

16. A composition comprising matrix/hepatic cell clusters grown on a three-dimensional support matrix wherein said matrix hepatic cell clusters comprising hepatocytes and nonparenchymal cells bound to a matrix coated with at least one biologically active molecule that promotes cell adhesion, proliferation or survival.

17. A three-dimensional tissue culture matrix prepared by a process comprising:

contacting a three-dimensional support matrix with a hepatic cell culture comprising hepatocytes and nonparenchymal cells bound to a matrix coated with at least one biologically active molecule that promotes cell adhesion, proliferation or survival;

under conditions sufficient to allow for the proliferation of the hepatic cell culture.

19. The method of claim 17 wherein the liver disorder is cirrhosis of the liver.

18. A method for providing hepatic function in a subject having a liver disorder comprising administering to said subject a three-dimensional tissue culture matrix prepared by a process comprising:

contacting a three-dimensional support matrix with a hepatic cell culture comprising hepatocytes and nonparenchymal cells bound to a matrix coated with at least one biologically active molecule that promotes cell adhesion, proliferation or survival, under conditions sufficient to allow for the proliferation of the hepatic cell culture;

in an amount sufficient to reduce the symptoms associated with the liver disorder.

20. The method of claim 18 wherein the liver disorder is hepatitis.