Cells or tissues with increased protein factors and methods of making and using same

• US 6,291,240 B1
• Filed: 08/21/1998
• Issued: 09/18/2001
• Est. Priority Date: 01/29/1998
• Status: Expired due to Term

• Current Assignee: Organogenesis Inc. (Organogenesis Holdings, Inc.)
• Original Assignee: Advanced Tissue Sciences, Inc.
• Inventors: Liu, Kang, Mansbridge, Jonathan N.
• Primary Examiner(s): Clark, Deborah J. R.
• Assistant Examiner(s): Kerr, Janet M.
• Application Number:
  US09/137,567
• Time in Patent Office: 1,124 Days
• Field of Search: 435/1.3, 435/325, 435/347, 435/375, 435/395, 435/402, 435/455, 435/240.23, 435/373, 536/23.1, 536/23.5, 536/24.1
• US Class Current: 435/395
• 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 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61L 27/22 Polypeptides or derivatives thereof , e.g. degradation products
• 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: C12N 2502/1323 Adult fibroblasts
• CPC Class Codes: C12N 2533/30 Synthetic polymers thermoreactive polymers, e.g. PNIPAm, C12N2539/10
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06

Abstract

The invention relates to cells or tissues having an increased amount of regulatory proteins, including cytokines, growth factors, angiogenic factors and/or stress proteins, and methods of producing and using those cells or tissues. The invention is based on the discovery that the production of regulatory proteins is induced in cells or tissue constructs following cryopreservation and subsequent thawing of the cells or constructs. The compositions and methods of this invention are useful for the treatment of wound healing and the repair and/or regeneration of other tissue defects including those of skin, cartilage, bone, and vascular tissue as well as for enhancing the culture and/or differentiation of cells and tissues in vitro.

Claims

1. A method for inducing the production of at least one regulatory protein selected from the group consisting of PDGF A, VEGF, TGF-β

1, bFGF, KGFA, HGF and HBEGF, in cells in vitro comprising;

(a) inoculating cells onto a substrate;

(b) culturing the cells on the substrate; and

(c) inducing the production of said at least one regulatory protein by subjecting the cells to cryopreservation and subsequent rapid thawing of between about 1 to about 3 minutes.

3. The method of claim 1 or 2 in which the cells are transfected with an exogenous nucleic acid under the control of an expression control element.

4. The method of claim 3 in which the expression control element comprises an inducible promoter.

6. The method of claim 1, 2 or 5 in which thawing is carried out in a water bath at approximately 34-37°

C.

2. A method for inducing the production of at least one regulatory protein selected from the group consisting of PDGF A, VEGF, TGF-β

1, bFGF, KGF, HGF and HBEGF, in a three-dimensional tissue construct in vitro comprising;

(a) inoculating stromal cells onto a three-dimensional biocompatible, non-living framework;

(b) culturing the cells in a nutrient environment to allow the cells to attach to and substantially envelop the framework to form a living stromal matrix on the framework thereby providing a three-dimensional tissue construct; and

(c) inducing the production of said at least one regulatory protein by subjecting the construct to cryopreservation and subsequent rapid thawing of between about 1 to about 3 minutes.

5. The method of claim 2, further comprising culturing parenchymal cells on the living stromal matrix, before or after induction of said regulatory protein.

7. The method of claim 5 in which the parenchymal cells are keratinocytes, hepatic, kidney, pancreatic, neuronal, or intestinal cells.

8. The method of claim 2 or 5 in which the stromal cells are mesenchymal stem cells, fibroblast-like cells, fibroblasts, chondro-progenitor cells, chondrocytes or any combination thereof.

9. The method of claim 5 in which the parenchymal cells are transfected with an exogenous nucleic acid under the control of an expression control element.

10. The method of claim 5 in which the stromal cells are transfected with an exogenous nucleic acid under the control of an expression control element.

11. The method of claim 5 in which both the stromal and parenchymal cells are transfected with at least one exogenous nucleic acid under the control of an expression control element.

12. The method of claim 9, 10 or 11 in which the expression control element comprises an inducible promoter.

13. The method of claim 12 in which the promoter is from the gene encoding a TGF-β

, VEGF, PDGF, KGF or basic FGF.

14. The method of claim 2 or 5 in which the framework is composed of a biodegradable material.

15. The method of claim 14 in which the biodegradable material is cotton, polyglycolic acid, a polycaprolactone, cat gut sutures, cellulose, gelatin, or dextran.

16. The method of claim 2 or 5 in which the framework is composed of a non-biodegradable material.

17. The method of claim 16 in which the non-biodegradable material is a polyamide, polyester, a polystyrene, a polypropylene, a polyacrylate, a polyvinyl, a polycarbonate, a polytetrafluoroethylene, or a nitrocellulose compound.

18. The method of claim 2 or 5 in which the framework is pre-coated with collagen.

19. The method of claim 2 or 5 in which the framework is a mesh.

20. A method for culturing parenchymal cells on a three-dimensional tissue construct prepared in vitro, wherein the tissue construct comprises a living stromal matrix 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, said tissue construct having been subjected to cryopreservation and subsequent rapid thawing of between about 1 to about 3 minutes and having an increased amount of at least one regulatory protein selected from the group consisting of PDGF A, VEGF, TGF-β

1, bFGF, KGF, HGF and HBEGF, relative to constructs that are not subjected to said cryopreservation and subsequent rapid thawing;

the method comprising;

(a) inoculating parenchymal cells onto the tissue construct; and

(b) incubating the inoculated tissue construct in a nutrient environment so that the inoculated cells proliferate in culture.

21. A method for culturing parenchymal cells in vitro comprising:

(a) inoculating parenchymal cells onto a three-dimensional tissue construct prepared in vitro, wherein the tissue construct comprises a living stromal matrix comprising stromal cells and connective tissue proteins naturally secreted by the stromal cells attached to and substantially envelopinga framework composed of a biocompatible, non-living material formed into a three-dimensional structure,

(b) subjecting the inoculated construct to cryopreservation and subsequent rapid thawing of between about 1 to about 3 minutes such that the inoculated construct contains an increased amount of at least one regulatory protein selected from the group consisting of PDGF A, VEGF, TGF-β

1, bFGF, KGF, HGF and HBEGF, relative to inoculated constructs that are not subjected to said cryopreservation and subsequent thawing and

(c) incubating the inoculated tissue construct in a nutrient environment so that the inoculated cells proliferate in culture.