Autogenic living scaffolds and living tissue matrices: methods and uses thereof

• US 10,226,548 B2
• Filed: 02/18/2015
• Issued: 03/12/2019
• Est. Priority Date: 08/29/2003
• Status: Active Grant

• Current Assignee: Genesis Technologies Limited
• Original Assignee: Genesis Technologies Limited
• Inventors: Ahlfors, Jan-Eric W.
• Primary Examiner(s): Leavitt, Maria G
• Application Number:
  US14/625,552
• Publication Number:
  US 20150273112A1
• Time in Patent Office: 1,483 Days
• Field of Search: None
• CPC Class Codes: A61L 2300/64 Animal cells
• CPC Class Codes: A61L 2430/00 Materials or treatment for tissue regeneration
• CPC Class Codes: A61L 2430/06 for cartilage reconstruction, e.g. meniscus
• CPC Class Codes: A61L 2430/28 for liver reconstruction
• CPC Class Codes: A61L 2430/30 for muscle reconstruction
• CPC Class Codes: A61L 2430/32 for nerve reconstruction
• CPC Class Codes: A61L 27/3633 Extracellular matrix [ECM]
• 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 27/3654 Cartilage, e.g. meniscus
• CPC Class Codes: A61L 27/367 Muscle tissue, e.g. sphincter
• CPC Class Codes: A61L 27/3675 Nerve tissue, e.g. brain, spinal cord, nerves, dura mater
• CPC Class Codes: A61L 27/3683 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
• CPC Class Codes: A61L 27/3687 characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
• 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 27/3834 Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem 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 27/54 Biologically active materials, e.g. therapeutic substances A61L27/227 takes precedence
• CPC Class Codes: A61L 27/56 Porous materials, e.g. foams or sponges
• CPC Class Codes: C12N 2513/00 : 3D culture
• CPC Class Codes: C12N 2533/90 : Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0619 : Neurons
• CPC Class Codes: C12N 5/0658 : Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
• CPC Class Codes: C12N 5/0671 : Three-dimensional culture, tissue culture or organ culture; Encapsulated cells

Abstract

The present invention is drawn to a 3-dimensional cell-produced scaffold construct comprising cells and the extracellular matrix that has been produced and arranged by these cells.

Claims

1. A three-dimensional cell-produced scaffold construct comprising fibroblast cells, myofibroblast cells, stromal cells, or endothelial cells and extracellular matrix synthesized by said cells,

wherein the extracellular matrix is synthesized in vitro by said cells, wherein said cells were seeded at a concentration between 200,000 to greater than 1,000,000 cells/cm² in chemically defined cell culture media that promote proliferation and extracellular matrix synthesis of said cells, wherein the cells were incubated for about one week to about 10 weeks in the chemically defined cell culture media to produce a scaffold construct having

a thickness of greater than 100 μ

m; and

a porosity that enables cellular migration of human neuroprogenitor cells throughout the construct.

2. The construct of claim 1, wherein some of said cells are genetically engineered.

3. The construct of claim 1, wherein said construct promotes differentiation, dedifferentiation, transdifferentiation or any combination thereof, of cells or tissue either within or in contact with the construct in vitro or in vivo.

4. The construct of claim 1, wherein said construct promotes cell growth, proliferation, migration and/or acquisition of in vivo-like morphology or any combination thereof, either within or in contact with the construct in vitro or in vivo.

5. The construct of claim 1, wherein said construct is an ex vivo cell-produced tissue.

6. The construct of claim 1, wherein said construct provides mechanical support, structural support, nutritional support, developmental support, support for wound repair, or supports the growth or regeneration of cells, tissue, organs, or any combination thereof.

7. The construct of claim 1, wherein said construct begins attaching to a site of implantation within about 36 hours, and continues attaching for several days, weeks, or months depending on the site of implantation and degree of immune system activity at the site of implantation.

8. The construct of claim 1, wherein said construct further comprises additional cells, tissue or any combination thereof.

9. The construct of claim 1, wherein the chemically defined cell culture media comprises:

(a) 3;

1 ratio of DMEM (high glucose (4.5 g/L);

with L-glutamine and sodium pyruvate and Ham'"'"'s F12 medium with the addition of;

(b) 5 μ

g/ml insulin;

(c) 5 ng/ml selenious acid;

(d) 10<sup>−

4</sup> M ethanolamine;

(e) 150 μ

g/ml L-ascorbic acid phosphate magnesium salt n-hydrate;

(f) 2.5 ng/ml epidermal growth factor in 5 μ

g/ml human serum albumin;

(g) 5 ng/ml basic fibroblast growth factor;

(h) 1.0×

10<sup>−

7</sup> M dexamethasone;

(i) 2×

10<sup>−

10</sup> M L-3,3′

,5-triiodothyronine;

(j) 4×

10<sup>−

3</sup> M Glutamax™

;

(k) 1 μ

g/ml glutathione; and

(l) 1% penicillin/streptomycin/amphotericin B.

10. The construct of claim 9, wherein the construct has a failure strain of at least 0.2.

22. The construct of claim 8, wherein said additional cells or tissue or combination thereof is selected from the group consisting of:

(a) stem cells;

(b) progenitor cells;

(c) cells or tissue of a connective, epithelial, muscle, nerve or glandular origin; and

(d) cells of vascular and/or non-vascular organ origin selected from the group consisting of neuroblastomas, myoblasts, astrocytes, cardiomyocytes, skeletal muscle myoblasts, hepatocytes, chrondrocytes, osteoblasts, fibroblasts, keratinocytes, Schwan cells, nerve cells, glial cells, epithelial cells, endothelial cells, smooth muscle cells, skeletal muscle cells, cardiac muscle cells, stromal cells, mesanglial cells, mesenchymal cells, hematopoietic cells, dendritic cells, immune system cells, neural tissue, hepatic tissue, aortic tissue, venous tissue, capillary tissue, cartilage, bone, muscle, glands, and hair follicles.

11. A three-dimensional cell-produced scaffold construct comprising fibroblast cells, myofibroblast cells, stromal cells, or endothelial cells and extracellular matrix synthesized by said cells,

wherein the extracellular matrix is synthesized in vitro by said cells, wherein said cells were seeded at a concentration between 1,000 cells/mm³ to greater than 200,000 cells/mm³ in chemically defined cell culture media that promote proliferation and extracellular matrix synthesis of said cells, wherein the cells were incubated for about one week to about 10 weeks in the chemically defined cell culture media to produce a scaffold construct having

a thickness of greater than 100 μ

m; and

a porosity that enables cellular migration of human neuroprogenitor cells throughout the construct.

12. The construct of claim 11, wherein some of said cells are genetically engineered.

13. The construct of claim 11, wherein said construct promotes differentiation, dedifferentiation, transdifferentiation or any combination thereof, of cells or tissue either within or in contact with the construct in vitro or in vivo.

14. The construct of claim 11, wherein said construct promotes cell growth, proliferation, migration and/or acquisition of in vivo-like morphology or any combination thereof, either within or in contact with the construct in vitro or in vivo.

15. The construct of claim 11, wherein said construct is an ex vivo cell-produced tissue.

16. The construct of claim 11, wherein said construct provides mechanical support, structural support, nutritional support, developmental support, support for wound repair, or supports the growth or regeneration of cells, tissue, organs, or any combination thereof.

17. The construct of claim 11, wherein said construct begins attaching to a site of implantation within about 36 hours, and continues attaching for several days, weeks, or months depending on the site of implantation and degree of immune system activity at the site of implantation.

18. The construct of claim 11, wherein said construct further comprises additional cells, tissue or any combination thereof.

19. The construct of claim 11, wherein the chemically defined cell culture media comprises:

(a) 3;

1 ratio of DMEM (high glucose (4.5 g/L);

with L-glutamine and sodium pyruvate and Ham'"'"'s F12 medium with the addition of;

(b) 5 μ

g/ml insulin;

(c) 5 ng/ml selenious acid;

(d) 10<sup>−

4</sup> M ethanolamine;

(e) 150 μ

g/ml L-ascorbic acid phosphate magnesium salt n-hydrate;

(f) 2.5 ng/ml epidermal growth factor in 5 μ

g/ml human serum albumin;

(g) 5 ng/ml basic fibroblast growth factor;

(h) 1.0×

10<sup>−

7</sup> M dexamethasone;

(i) 2×

10<sup>−

10</sup> M L-3,3′

,5-triiodothyronine;

(j) 4×

10<sup>−

3</sup> M Glutamax™

;

(k) 1 μ

g/ml glutathione; and

(l) 1% penicillin/streptomycin/amphotericin B.

20. The construct of claim 19, wherein the construct has a failure strain of at least 0.2.

21. The construct of claim 18, wherein said additional cells or tissue or combination thereof is selected from the group consisting of:

(a) stem cells;

(b) progenitor cells;

(c) cells or tissue of a connective, epithelial, muscle, nerve or glandular origin; and

(d) cells of vascular and/or non-vascular organ origin selected from the group consisting of neuroblastomas, myoblasts, astrocytes, cardiomyocytes, skeletal muscle myoblasts, hepatocytes, chrondrocytes, osteoblasts, fibroblasts, keratinocytes, Schwan cells, nerve cells, glial cells, epithelial cells, endothelial cells, smooth muscle cells, skeletal muscle cells, cardiac muscle cells, stromal cells, mesanglial cells, mesenchymal cells, hematopoietic cells, dendritic cells, immune system cells, neural tissue, hepatic tissue, aortic tissue, venous tissue, capillary tissue, cartilage, bone, muscle, glands, and hair follicles.