Three-dimensional cell and tissue culture system

• US 5,032,508 A
• Filed: 09/01/1989
• Issued: 07/16/1991
• Est. Priority Date: 09/08/1988
• 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): Kepplinger, Esther L.
• Assistant Examiner(s): SCHEINER, TONI R
• Application Number:
  US07/402,104
• Time in Patent Office: 683 Days
• Field of Search: 435/29, 435/32, 435/1, 435/244, 435/240.23, 435/240.243, 436/63
• US Class Current: 435/32
• 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: A61L 27/38 containing added animal cells organs or tissue containing native cells A61L27/36
• CPC Class Codes: A61L 27/60 Materials for use in artificial skin
• CPC Class Codes: C12N 2502/081 neurons
• CPC Class Codes: C12N 2502/086 glial cells
• CPC Class Codes: C12N 2502/091 melanocytes
• CPC Class Codes: C12N 2502/094 keratinocytes
• CPC Class Codes: C12N 2502/097 oral mucosa cells
• CPC Class Codes: C12N 2502/1305 Adipocytes
• CPC Class Codes: C12N 2502/1323 Adult fibroblasts
• CPC Class Codes: C12N 2502/28 Vascular endothelial cells
• CPC Class Codes: C12N 2502/30 tumour cells
• 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 5/0062 General methods for three-dimensional culture
• CPC Class Codes: C12N 5/0618 Cells of the nervous system
• CPC Class Codes: C12N 5/0669 : Bone marrow stromal cells; Whole bone marrow isolated stem cells from bone marrow C12N5/0647, C12N5/0663
• CPC Class Codes: C12N 5/0671 : Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
• CPC Class Codes: C12N 5/0677 : Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
• CPC Class Codes: C12N 5/0697 : Artificial constructs associating cells of different lineages, e.g. tissue equivalents blood vessels C12N5/0691
• CPC Class Codes: C12N 5/0698 : Skin equivalents

Abstract

The present invention relates to a three-dimensional cell culture system which can be used to culture a variety of different cells and tissues in vitro for prolonged periods of time. In accordance with the invention, cells derived from a desired tissue are inoculated and grown on a pre-established stromal support matrix. The stromal support matrix comprises stromal cells, such as fibroblasts actively growing on a three-dimensional matrix. Stromal cells may also include other cells found in loose connective tissue such as endothelial cells, macrophages/monocytes, adipocytes, pericytes, reticular cells found in bone marrow stroma, etc. The stromal matrix provides the support, growth factors, and regulatory factors necessary to sustain long-term active proliferation of cells in culture. When grown in this three-dimensional system, the proliferating cells mature and segregate properly to form components of adult tissues analogous to counterparts found in vivo.

Claims

1. A method for testing the cytological effect of a test substance comprising:

(a) exposing a three dimensional cell culture to the test substance, in which the three dimensional cell culture comprises 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 a biocompatible, non-living material formed into a three dimensional structure having interstitial spaces bridges by the stromal cells; and

(b) determining the effect of the test substance by measuring a change in the three dimensional cell culture.

2. The method of claim 1 for testing the cytotoxicity of a test substance, wherein the effect of the test substance is determined by measuring the number of damaged or deed cells in the three dimensional cell culture, in which the number of damaged or dead cells correlates with the cytotoxicity of the test substance.

3. The method of claim 1 for testing the growth/regulatory activity of a test substance, wherein the effect of the test substance is determined by analyzing the cellular content of the three dimensional cell culture, in which an increase in cell number or a cell type correlates with the growth/regulatory activity of the test substance.

4. The method of claim 1 for testing the metabolic activity of a test substance, wherein the effect of the test substance is determined by measuring a metabolite produced by the three dimensional cell culture, in which an increase or decrease in the amount of metabolite produced correlates with the activity of the test substance.

5. The method of claim 1 for testing the allergenic effect of a test substance, wherein the three dimensional cell cultures containing mast cells, and the effect of the test substance is determined by measuring the release of a vasoactive mediator by the mast cells, in which release of the vasoactive mediator correlates with the allergenic effect of the test substance.

6. The method for testing the cytological effect of a test substance according to claim 1 in which the stromal cells are fibroblasts.

7. The method for testing the cytological effect of a test substance according to claim 1 in which the stromal cells are a combination of fibroblasts and endothelial cells, perictyes, macrophages, monocytes, leukocytes, plasma cells, mast cells or adipocytes.

8. The method for testing the cytological effect of a test substance according to claim 1 in which the framework is composed of a biodegradable material.

9. The method for testing the cytological effect of a test substance according to claim 8 in which the biodegradable material is cotton, polyglycolic acid, cat gut sutures, cellulose, gelatin, or dextran.

10. The method for testing the cytological effect of a test substance according to claim 1 in which the framework is composed of a non-biodegradable material.

11. The method for testing the cytological effect of a test substance according to claim 10 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.

12. The method for testing the cytological effect of a test substance according to claim 8, 9, 10 or 11 in which the framework is pre-coated with collagen.

13. The method for testing the cytological effect of a test substance according to claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 in which the framework is a mesh.

14. The method for testing the cytological effect of a test substance according to claim 12 in which the framework is a mesh.

15. The method for testing the cytological effect of a test substance according to claim 1 in which the parenchymal cells are hematopoietic cells.

16. The method for testing the cytological effect of a test substance according to claim 1 in which the parenchymal cells are melanocytes and keratinocytes.

17. The method for testing the cytological effect of a test substance according to claim 1 in which the parenchymal cells are hepatocytes.

18. The method for testing the cytological effect of a test substance according to claim 11 in which the parenchymal cells are pancreatic acinar cells.

19. The method for testing the cytological effect of a test substance according to claim 1 in which the parenchymal cells are kidney cells.