Methods of use of a three-dimensional liver cell and tissue culture system

• US 5,849,588 A
• Filed: 08/01/1996
• Issued: 12/15/1998
• Est. Priority Date: 04/18/1986
• Status: Expired due to Term

• Current Assignee: RegeneMed, Inc.
• Original Assignee: Advanced Tissue Sciences, Inc.
• Inventors: Naughton, Gail K., Naughton, Brian A.
• Primary Examiner(s): Saucier, Sandra E.
• Application Number:
  US08/690,955
• Time in Patent Office: 866 Days
• Field of Search: 435/240, 435/243, 435/240.1, 435/240.2, 435/240.23, 435/284.1, 435/395, 435/396, 435/397, 435/398, 435/399, 435/400, 435/402, 435/29, 435/347, 435/373
• US Class Current: 435/373
• CPC Class Codes: A61B 2010/0258 Marrow samples
• CPC Class Codes: A61B 90/00 Instruments, implements or accessories specially adapted for surgery or diagnosis and not covered by any of the groups A61B1/00 - A61B50/00, e.g. for luxation treatment or for protecting wound edges protective face masks A41D13/11; surgeon's or patient's gowns or dresses A41D13/12; devices for carrying-off, for treatment of, or for carrying-over, body liquids A61M1/00
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• 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: A61K 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• CPC Class Codes: A61L 27/3604 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
• CPC Class Codes: A61L 27/3629 Intestinal tissue, e.g. small intestinal submucosa
• CPC Class Codes: A61L 27/3637 characterised by the origin of the biological material other than human or animal, e.g. plant extracts, algae
• CPC Class Codes: A61L 27/3641 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: 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: A61L 27/3886 comprising two or more cell types
• CPC Class Codes: A61L 27/3895 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
• CPC Class Codes: A61L 27/60 Materials for use in artificial skin
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/33 Insulin together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• CPC Class Codes: C12N 2501/335 Glucagon; Glucagon-like peptide [GLP]; Exendin
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2503/04 : Screening or testing on artificial tissues
• CPC Class Codes: C12N 2510/00 : Genetically modified cells
• CPC Class Codes: C12N 2533/30 : Synthetic polymers thermoreactive polymers, e.g. PNIPAm, C12N2539/10
• CPC Class Codes: C12N 2533/40 : Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
• CPC Class Codes: C12N 2533/70 : Polysaccharides
• CPC Class Codes: C12N 5/0671 : Three-dimensional culture, tissue culture or organ culture; Encapsulated cells

Abstract

The present invention relates to a three-dimensional cell and tissue culture system. In particular, it relates to this culture system for the long term culture of liver cells and tissues in vitro in an environment that more closely approximates that found in vivo. The culture system described herein provides for proliferation and appropriate liver cell maturation to form structures analogous to tissue counterparts in vivo. The resulting liver tissues survive for prolonged periods, perform liver-specific functions, and maintain hepatic tissue architecture following in vivo implantation.

The liver cultures have a variety of applications ranging from transplantation or implantation in vivo, to screening cytotoxic compounds and pharmaceutical compounds in vitro, to the production of biologically active molecules in "bioreactors" and to the construction of extracorporeal liver assist device.

Claims

1. A method for determining the effect of a drug on liver parenchymal cells in culture, comprising:

(a) exposing a three-dimensional liver cell culture to the drug, in which the three dimensional cell culture comprises liver parenchymal cells grown on a living stromal tissue prepared in vitro, comprising stromal cells and connective tissue proteins naturally secreted by the stromal cells attached to and substantially enveloping a framework composed of biocompatible, non-living material formed into a three dimensional structure having interstitial spaces bridged by the stromal cells; and

(b) determining the effect of the drug on the liver parenchymal cells in culture.

2. The method for testing the effect of a drug according to claim 1 in which the stromal cells are fibroblasts.

3. The method for testing the effect of a drug according to claim 1 in which the stromal cells are a combination of fibroblasts and endothelial cells, pericytes, macrophages, monocytes, leukocytes, plasma cells, mast cells or adipocytes.

4. The method for testing the effect of a drug according to claim 1 in which the framework is composed of a biodegradable material.

5. The method for testing the effect of a drug according to claim 4 in which the biodegradable material is cotton, polyglycolic acid, cat gut sutures, cellulose, gelatin, or dextran.

6. The method for testing the effect of a drug according to claim 1 in which the framework is composed of a non-biodegradable material.

7. The method for testing the effect of a drug according to claim 6 in which the non-biodegradable material is a polyamide, a polyester, a polystyrene, a polypropylene, a polyacrylate, a polyvinyl, a polycarbonate, a polytetrafluorethylene, or a nitrocellulose compound.

8. The method for testing the effect of a drug according to claims 4, 5, 6 or 7 in which the framework is pre-coated with collagen.

9. The method according to claim 8 in which the framework is a mesh.

10. The method according to claims 1, 2, 3, 4, 5, 6, or 7 in which the framework is a mesh.

11. A method for studying the mechanism of a disease or condition in a patient, which disease or condition has a detectable effect on liver parenchymal cells in culture comprising:

(a) inoculating liver parenchymal cells from a sample obtained from the patient onto a living stromal tissue prepared in vitro, comprising stromal cells and connective tissue proteins naturally secreted by the stromal cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells;

(b) culturing the inoculated living stromal tissue in a nutrient medium so that the inoculated liver parenchymal cells proliferate in culture; and

(c) analyzing the proliferated liver parenchymal cells in culture for markers of the disease or condition.

12. The method for studying disease mechanisms according to claim 11 in which the stromal cells are fibroblasts.

13. The method for studying disease mechanisms according to claim 11 in which the stromal cells are a combination of fibroblasts and endothelial cells, pericytes, macrophages, monocytes, leukocytes, plasma cells, mast cells or adipocytes.

14. The method for studying disease mechanisms according to claim 11 in which the framework is composed of a biodegradable material.

15. The method for studying disease mechanisms according to claim 14 in which the biodegradable material is cotton, polyglycolic acid, cat gut sutures, cellulose, gelatin, or dextran.

16. The method for studying disease mechanisms according to claim 11 in which the framework is composed of a non-biodegradable material.

17. The method for studying disease mechanisms according to claim 11 in which the non-biodegradable material is a polyamide, a polyester, a polystyrene, a polypropylene, a polyacrylate, a polyvinyl, a polycarbonate, a polytetrafluorethylene, or a nitrocellulose compound.

18. The method for studying disease mechanisms according to claims 14, 15, 16, or 17 in which the framework is pre-coated with collagen.

19. The method according to claim 18 in which the framework is a mesh.

20. The method according to claims 11, 12, 13, 14, 15, 16 or 17 in which the framework is a mesh.