Three-dimensional skin cell and tissue culture system

• US 5,512,475 A
• Filed: 04/06/1995
• Issued: 04/30/1996
• Est. Priority Date: 04/18/1986
• Status: Expired due to Term

• Current Assignee: Shire Regenerative Medicine Incorporated (Shire plc)
• Original Assignee: Advanced Tissue Sciences, Inc.
• Inventors: Naughton, Gail K., Naughton, Brian A.
• Primary Examiner(s): Wityshyn, Michael G.
• Assistant Examiner(s): Williams, Jane
• Application Number:
  US08/418,230
• Time in Patent Office: 390 Days
• Field of Search: 435/1,29,32,240.243,240.1,240.2,284,240.23, 623/15,11, 424/424,484,422,572
• US Class Current: 424/484
• 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 27/3886 comprising two or more cell types
• 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 2502/091 melanocytes
• CPC Class Codes: C12N 2502/094 keratinocytes
• CPC Class Codes: C12N 2502/1323 Adult 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 5/0698 : Skin equivalents
• CPC Class Codes: Y10S 623/915 : Method or apparatus for preparing biological material

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 transplantation or implantation of a three-dimensional skin cell culture comprising implanting a culture of melanocyte and keratinocyte cells cultured on a living stromal tissue prepared in vitro, said living stromal tissue 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 so that a tissue equivalent is formed.

2. The method according to claim 1 in which the stromal cells are fibroblasts.

3. The method 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 according to claim 1 in which the framework is composed of a biodegradable material.

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

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

7. The method 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.

17. The method according to claim 1, 8, 2, 3, 9, 10, 4, 5, 11, 12, 6, 7, 13 or 14 in which the framework is a mesh.

8. A method for transplantation or implantation of a three-dimensional culture comprising implanting in vivo melanocytes and keratinocytes cultured on a living stromal tissue prepared in vitro, said living stromal tissue comprising confluent 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 so that a tissue equivalent is formed.

9. The method according to claim 8 in which the stromal cells are fibroblasts.

10. The method according to claim 8 in which the stromal cells are a combination of fibroblasts and endothelial cells, pericytes, macrophages, monocytes, leukocytes, plasma cells, mast cells or adipocytes.

11. The method according to claim 8 in which the framework is composed of a biodegradable material.

12. The method according to claim 11 in which the biodegradable material is cotton, polyglycolic acid, cat gut sutures, cellulose, gelatin, or dextran.

13. The method according to claim 8 in which the framework is composed of a non-biodegradable material.

14. The method according to claim 13 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.

15. The method according to claim 4, 5, 11, 12, 6, 7, 13 or 14 in which the framework is pre-coated with collagen.

16. The method according to claim 15 in which the framework is a mesh.

18. A method for determining the effect of a drug on melanocyte and keratinocyte cells in culture comprising:

(a) exposing a three-dimensional skin cell culture to the drug, in which the three-dimensional skin cell culture comprises melanocyte and keratinocyte cells grown on a living stromal tissue prepared in vitro, said living stromal tissue 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; and

(b) determining the effect of the drug on the melanocyte and keratinocyte cells in culture.

19. The method for determining the effect of a drug according to claim 18 in which the stromal cells are fibroblasts.

20. The method for determining the effect of a drug according to claim 18 in which the stromal cells are a combination of fibroblasts and endothelial cells, pericytes, macrophages, monocytes, leukocytes, plasma cells, mast cells or adipocytes.

21. The method for determining the effect of a drug according to claim 18 in which the framework is composed of a biodegradable material.

22. The method for determining the effect of a drug according to claim 21 in which the biodegradable material is cotton, polyglycolic acid, cat gut sutures, cellulose, gelatin, or dextran.

23. The method for determining the effect of a drug according to claim 18 in which the framework is composed of a non-biodegradable material.

24. The method for determining the effect of a drug according to claim 23 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.

25. The method for determining the effect of a drug according to claim 21, 22, 23 or 24 in which the framework is pre-coated with collagen.

26. The method according to claim 25 in which the framework is a mesh.

27. The method according to claim 18, 19, 20, 21, 22, 23 or 24 in which the framework is a mesh.

28. A method for studying the mechanisms of a disease or condition in a patient, which disease or condition has a detectable affect on melanocyte and keratinocyte cells in culture, comprising:

(a) inoculating melanocyte and keratinocyte cells obtained from the patient onto a living stromal tissue prepared in vitro, said living stromal tissue 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) incubating the inoculated living stromal tissue in a nutrient medium so that the inoculated melanocyte and keratinocyte cells proliferate in culture; and

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

29. The method according to claim 28 in which the stromal cells are fibroblasts.

30. The method according to claim 28 in which the stromal cells are a combination of fibroblasts and endothelial cells, pericytes, macrophages, monocytes, leukocytes, plasma cells, mast cells or adipocytes.

31. The method according to claim 28 in which the framework is composed of a biodegradable material.

32. The method according to claim 31 in which the biodegradable material is cotton, polyglycolic acid, cat gut sutures, cellulose, gelatin, or dextran.

33. The method according to claim 28 in which the framework is composed of a non-biodegradable material.

34. The method according to claim 33 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.

35. The method according to claim 31, 32, 33 or 34 in which the framework is pre-coated with collagen.

36. The method according to claim 35 in which the framework is a mesh.

37. The method according to claim 28, 29, 30, 31, 32, 33 or 34 in which the framework is a mesh.