Three-dimensional cartilage cultures

• US 5,902,741 A
• Filed: 06/05/1995
• Issued: 05/11/1999
• Est. Priority Date: 04/18/1986
• Status: Expired due to Term

• Current Assignee: Smith & Nephew Incorporated (Smith & Nephew PLC)
• Original Assignee: Advanced Tissue Sciences, Inc.
• Inventors: Purchio, Anthony F., Zimber, Michael, Dunkelman, Noushin, Naughton, Gail K., Naughton, Brian A.
• Primary Examiner(s): Stucker, Jeffrey
• Assistant Examiner(s): PARK, HANKYEL
• Application Number:
  US08/463,566
• Time in Patent Office: 1,436 Days
• Field of Search: 435/1.1, 435/240.2, 424/572, 424/574, 623/15
• US Class Current: 435/325
• 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: A61F 2/0063 Implantable repair or support meshes, e.g. hernia meshes
• CPC Class Codes: A61F 2/04 Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts A61F2/18, A61F2/20 take precedence; devices, other than stent-grafts, providing patency to, or preventing collapsing of, tubular structures of the body other than stent-grafts, e.g. stents A61F2/82; instruments specially adapted for placement or removal of stents or stent-grafts A61F2/95
• CPC Class Codes: A61F 2/06 Blood vessels
• CPC Class Codes: A61F 2/08 Muscles; Tendons; Ligaments sutures A61B17/04
• 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 2430/06 for cartilage reconstruction, e.g. meniscus
• 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 27/3852 Cartilage, e.g. meniscus
• 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 2501/15 : Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/33 : Insulin
• CPC Class Codes: C12N 2501/335 : Glucagon; Glucagon-like peptide [GLP]; Exendin
• CPC Class Codes: C12N 2501/39 : Steroid hormones
• 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 2533/40 : Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
• CPC Class Codes: C12N 5/0655 : Chondrocytes; Cartilage
• CPC Class Codes: G01N 2500/00 : Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 33/5088 : of vertebrates

Abstract

The present invention relates to a method of stimulating the proliferation and appropriate cell maturation of a variety of different cells and tissues in three-dimensional cultures in vitro using TGF-β in the culture medium. In accordance with the invention, stromal cells, including, but not limited to, chondrocytes, chondrocyte-progenitors, fibroblasts, fibroblast-like cells, umbilical cord cells or bone marrow cells from umbilical cord blood are inoculated and grown on a three-dimensional framework in the presence of TGF-β. 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 cells and connective tissue proteins naturally secreted by the stromal cells attach to and substantially envelope the framework composed of a biocompatible non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells. The living stromal tissue so formed provides the support, growth factors, and regulatory factors necessary to sustain long-term active proliferation of cells in culture and/or cultures implanted in vivo. When grown in this three-dimensional system, the proliferating cells mature and segregate properly to form components of adult tissues analogous to counterparts in vivo.

Claims

1. A method for preparing a living cartilage tissue in vitro, comprising culturing cartilage-producing stromal cells inoculated onto a three-dimensional framework in a nutrient medium, so that the stromal cells and connective tissue proteins naturally secreted by the stromal cells attach to and substantially envelope the framework composed of a biocompatible, non-living material formed into a three dimensional structure having interstitial spaces bridged by the stromal cells to form into a three-dimensional cartilage construct.

2. The method of claim 1 in which the stromal cells are chondrocytes or chondrocyte-progenitor cells.

3. The method of claim 1 in which the stromal cells are fibroblasts or fibroblast-like cells.

4. The method of claim 1 in which the stromal cells are umbilical cord cells or bone marrow cells from umbilical cord blood.

5. The method of claim 1 in which the cells comprise a combination of:

chondrocytes, chondrocyte-progenitors, fibroblasts, fibroblast-like cells, endothelial cells, pericytes, macrophages, monocytes, leukocytes, plasma cells, mast cells, adipocytes, umbilical cord cells, placental, or bone marrow cells from umbilical cord blood.

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

7. The method of claim 6 in which the biodegradable material is polyglycolic acid, cotton, cat gut sutures, cellulose, gelatin, collagen or polyhydroxyalkanoates.

8. The method of claim 1 in which the framework is treated with ethylene oxide.

9. The method of claim 1 in which the framework is treated with an electron beam.

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

11. The method of 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 of claim 1 in which the framework is a mesh.

13. The method of claim 1 in which the culture medium further comprises of an effective amount of ascorbate.

14. The method of claim 1 in which the culture medium is kept under static conditions.

15. The method of claim 1 in which the culture medium is kept in dynamic state by convection and under periodic pressurization.

28. The method of claim 1, 2, 3, 4 or 5 in which the stromal cells are transfected with an exogenous gene under the control of an expression element.

30. The method of claim 2 or 5 in which the chondrocyte-progenitor cells are mesenchymal stem cells.

32. The method of claim 1 in which the nutrient medium contains a growth factor in an amount effective to enhance the stromal cell production of cartilage matrix proteins.

33. The method of claim 32 in which the growth factor is TGF-β

.

34. The method of claim 1 in which the stromal cells are cultured on the framework in a bioreactor.

16. A living cartilage tissue prepared in vitro, comprising cartilage-producing stromal cells and connective tissue proteins naturally secreted by the stromal cells attached to and substantially enveloping a three-dimensional framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells.

17. The living cartilage tissue of claim 16 in which the stromal cells are chondrocytes or chondrocyte-progenitor cells.

18. The living cartilage tissue of claim 16 in which the stromal cells are fibroblasts or fibroblast-like cells.

19. The living cartilage tissue of claim 16 in which the stromal cells are umbilical cord cells, placental cells or bone marrow cells from umbilical cord blood.

20. The living cartilage tissue of claim 16 in which the framework was treated with ethylene oxide.

21. The living cartilage tissue of claim 16 in which the framework was treated with an electron beam.

22. The living cartilage tissue of claim 16 in which the framework is composed of a biodegradable material.

23. The living cartilage tissue of claim 22 in which the biodegradable material is polyglycolic acid, cotton, cat gut sutures, cellulose, gelatin, collagen or polyhydroxyalkanoates.

24. The living cartilage tissue of claim 16 in which the framework is composed of a non-biodegradable material.

25. The living cartilage tissue of claim 24 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.

26. The living cartilage tissue of claim 16 in which the framework is a mesh or a sponge.

27. The living cartilage tissue of claim 16 in which the cartilage-producing stromal cells comprise a combination of:

chondrocytes, chondrocyte-progenitors, fibroblasts, fibroblast-like cells, muscle cells, umbilical cord cells, placental cells or bone marrow cells from umbilical cord blood.

29. The living cartilage tissue of claim 16, 17, 18, 19, or 27 in which the stromal cells are transfected with an exogenous gene under the control of an expression element.

31. The living cartilage tissue of claim 19 or 27 in which the chondrocyte-progenitor cells are mesenchymal stem cells.

35. A composition for growing new cartilage comprising mesenchymal stem cells in a polymeric carrier suitable for proliferation and differentiation of the cells into cartilage.

36. The composition of claim 35 wherein the mesenchymal stem cells are isolated from muscle or dermis.