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

• US 8,043,614 B2
• Filed: 03/09/2005
• Issued: 10/25/2011
• Est. Priority Date: 03/09/2004
• Status: Active Grant

• Current Assignee: Genesis Technologies Limited
• Original Assignee: Jan-Eric W. Ahlfors
• Inventors: Ahlfors, Jan-Eric W.
• Primary Examiner(s): LEAVITT, MARIA GOMEZ
• Application Number:
  US11/077,185
• Publication Number:
  US 20050226856A1
• Time in Patent Office: 2,421 Days
• Field of Search: None
• US Class Current: 424/93.7
• 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 fibroblasts and the extracellular matrix that has been produced and arranged by these cells. The constructs promote the differentiation, dedifferentiation and/or transdifferentiation of cells and/or formation of tissue in vitro and in vivo, while at the same time promoting cell growth, proliferation, migration, and acquisition of in vivo-like morphology.

Claims

1. A three-dimensional cell-produced scaffold construct comprising extracellular matrix synthesized in vitro by fibroblast cells, wherein said construct is produced by a process comprising:

i) incubating the fibroblast cells at a concentration of at least about 200,000 cells/cm² in defined chemical media containing components that promote proliferation and extracellular matrix synthesis of said fibroblast cells; and

ii) isolating the construct from the media,

wherein the cells are incubated for a period of time in the media that is sufficient to produce a construct having a thickness and porosity that enables cellular migration of additional cells throughout the construct wherein the construct has a failure strain of at least about 0.2.

2. The construct of claim 1, wherein the fibroblast cells are seeded at a concentration of at least about 200,000 cells/cm².

3. The construct of claim 1, wherein the defined chemical media is serum-free media comprising base media, basic fibroblast growth factor, epidermal growth factor, dexamethasone, L-3,3′

,5-triiodothyronine, insulin, selenious acid, and a lipid precursor.

4. The construct of claim 1, wherein said construct has a tensile strength of at least 200 kilopascals within three weeks of growth.

5. The construct of claim 3, wherein the base media is selected from the group consisting of Dulbecco'"'"'s Modification of Eagle'"'"'s Medium (DMEM);

Ham'"'"'s F-12 (F12);

Ham'"'"'s F-10 (F10);

Iscove'"'"'s Modification of DMEM (IDMEM);

MEDIUM 199;

Alpha MEM;

Basal Medium Eagle with Earle'"'"'s BSS;

DMEM;

F1.2 1;

1 with L-Glutamine;

Glasgow'"'"'s MEM with L-glutamine (GMEM);

IMDM with HEPES, with or without L-Glutamine;

L-15, without L-Glutamine;

McCoy'"'"'s 5A Modified Medium;

Medium 199;

MEM Eagle;

MEM Eagle-Earle'"'"'s BSS, with or without L-Glutamine;

MEM Eagle-Flanks BSS;

NCTC-109;

Richter'"'"'s CM Medium;

RPMI 1640 with HEPES;

RPMI 1640 and a combination thereof.

6. The construct of claim 5, wherein the serum-free defined chemical media further comprises additional components selected from the group consisting of platelet derived growth factor (PDGF), L-cysteine, Trolox and combinations thereof.

7. The construct of claim 3, wherein the serum-free defined chemical media comprises:

(i) L-ascorbic acid phosphate magnesium salt n-hydrate in a concentration range of from 0.1 mM to 3 mM;

(ii) epidermal growth factor in a concentration range of from 0.002 nM to 2 nM;

(iii) basic fibroblast growth factor in a concentration range of from 0.03 nM to over 3 nM;

(iv) insulin in a concentration range of from 10 μ

M to 1000 μ

M;

(v) L-3,3′

,5-triiodothyronine in a concentration range of from 0.1 nM to 10 nM;

(vi) ethanolamine in a concentration range of from 1 μ

M to 10,000 μ

M;

(vii) selenious acid in a concentration range of from 10 mM to 1000 mM;

(viii) Glutamax in a concentration range of from 0 mM to 10 mM; and

(ix) dexamethasone in a concentration range of from 1 nM to 1000 nM.

8. The construct of claim 3, wherein the serum-free defined chemical 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 supplemented with the following components;

(i) 4.2×

10<sup>−

10</sup> M epidermal growth factor;

(ii) 2.8×

10<sup>−

10</sup> M basic fibroblast growth factor

(iii) 8.6×

10<sup>−

5</sup> M insulin

(iv) 1.0×

10<sup>−

7</sup> M dexamethasone

(v) 3.2×

10<sup>−

4</sup> M L-ascorbic acid phosphate magnesium salt n-hydrate

(vi) 2×

10<sup>−

10</sup> L-3,3′

,5-triiodothyronine

(vii) 10<sup>−

4</sup> M ethanolamine

(viii) 3.9×

10<sup>−

8</sup> M selenious acid

(ix) 4×

10<sup>−

3</sup> M Glutamax

(x) 3.3×

10<sup>−

6</sup> M glutathione (reduced); and

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

9. The construct of claim 8, wherein the serum-free defined chemical media further comprises an additional component selected from the group consisting of platelet derived growth factor in a concentration of from 0.1 ng/ml to 10 ng/ml;

L-cysteine in a concentration range of from 0.1 mM to 1 mM, and combinations thereof.

10. The construct of claim 1, wherein the fibroblast cells are seeded at a density of between 200,000 to 1,000,000 cells/cm².

11. The construct of claim 3, wherein the medium is replaced every 2-3 days.

12. The construct of claim 1, wherein the construct has a growth rate in the media such that it is capable of achieving a thickness of greater than 100 μ

m within three weeks of growth.

24. The construct of claim 1, wherein said construct:

(a) is seeded with additional cells that grow and are integrated three dimensionally throughout said construct;

(b) provides mechanical support for said additional cells; and

(c) provides components for development of said additional cells.

26. The construct of any one of claim 1, 13, 14, 24 or 25, wherein said fibroblast cells are maintained at a density of about 1,000 cells/mm³ to greater than about 200,000 cells/mm³.

27. The construct of any one of claim 1, 13, 14, 24 or 25, wherein said construct further comprises additional cells, tissue or any combination thereof.

28. The construct of any one of claim 1, 13, 14, 24 or 25, 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.

29. The construct of any one of claim 1, 13, 14, 24 or 25, 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.

30. The construct of any one of claim 1, 13, 14, 24 or 25, wherein said construct is an ex vivo cell-produced tissue.

31. The construct of claim 24, wherein said additional cells, tissue or combination thereof, comprise:

(a) stem cells;

(b) progenitor cells;

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

(d) cells of the 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.

32. The construct of any one of claim 1, 13, 14, 24 or 25, 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.

33. The construct of any one of claim 1, 13, 14, 24 or 25, wherein said construct comprises neural cells.

34. The construct of claim 33, wherein the lifespan of said neural cells is extended at least about two-fold compared to neural cells in the absence of the construct.

35. The construct of claim 33, wherein the fragility of said neural cells is reduced compared to neural cells in the absence of said construct.

36. The construct of any one of claim 1, 13, 14, 24 or 25, 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.

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

39. The construct of claim 1, wherein said cellular migration throughout said construct comprises migration of cells from a subject into said construct upon implantation thereof into said subject.

40. The construct of claim 27, wherein said additional cells, tissue or combination thereof, comprise:

(a) stem cells;

(b) progenitor cells;

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

(d) cells of the 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.

13. A three-dimensional cell-produced scaffold construct comprising fibroblast cells and extracellular matrix synthesized in vitro by said fibroblast cells, wherein said construct has a thickness and porosity that enables cellular migration throughout the construct, wherein the construct has a growth rate in the media such that it is capable of achieving a thickness of greater than 100 μ

m within three weeks of growth, wherein the construct is made by a process comprising;

i) incubating the fibroblast cells at a concentration of at least about 200,000 cells/cm² in defined chemical media containing components that promote proliferation and extracellular matrix synthesis of said fibroblast cells; and

ii) isolating the construct from the media.

15. The construct of claim 13, wherein the fibroblast cells are seeded at a concentration of at least 200,000 cells/cm² in serum-free defined chemical media.

16. The construct of claim 15, wherein the serum-free defined chemical media comprises base media, basic fibroblast growth factor, epidermal growth factor, dexamethasone, L-3,3′

,5-triiodothyronine, insulin, selenious acid, and a lipid precursor.

17. The construct of claim 16, wherein the base media is selected from the group consisting of Dulbecco'"'"'s Modification of Eagle'"'"'s Medium (DMEM);

Ham'"'"'s F-12 (F12);

Ham'"'"'s F-10 (F10);

Iscove'"'"'s Modification of DMEM (IDMEM);

MEDIUM 199;

Alpha MEM;

Basal Medium Eagle with Earle'"'"'s BSS;

DMEM;

F12 1;

1 with L-Glutamine;

Glasgow'"'"'s MEM with L-glutamine (GMEM);

IMDM with HEPES, with or without L-Glutamine;

L-15, without L-Glutamine;

McCoy'"'"'s 5A Modified Medium;

Medium 199;

MEM Eagle;

MEM Eagle-Earle'"'"'s BSS, with or without L-Glutamine;

MEM Eagle-Hanks BSS;

NCTC-109;

Richter'"'"'s CM Medium;

RPMI 1640 with HEPES;

RPMI 1640 and a combination thereof.

18. The construct of claim 17, wherein the serum-free defined chemical media further comprises additional components selected from the group consisting of platelet derived growth factor (PDGF), L-cysteine, Trolox and combinations thereof.

19. The construct of claim 15, wherein the serum-free defined chemical media comprises:

(i) L-ascorbic acid phosphate magnesium salt n-hydrate in a concentration range of from 0.1 mM to 3 mM;

(ii) epidermal growth factor in a concentration range of from 0.002 nM to 2 nM;

(iii) basic fibroblast growth factor in a concentration range of from 0.03 nM to over 3 nM;

(iv) insulin in a concentration range of from 10 μ

M to 1000 μ

M;

(v) L-3,3′

,5-triiodothyronine in a concentration range of from 0.1 nM to 10 nM;

(vi) ethanolamine in a concentration range of from 1 μ

M to 10,000 μ

M;

(vii) selenious acid in a concentration range of from 10 nM to 1000 nM;

(viii) Glutamax in a concentration range of from 0 mM to 10 mM; and

(ix) dexamethasone in a concentration range of from 1 nM to 1000 nM.

20. The construct of claim 15, wherein the serum-free defined chemical 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 supplemented with the following components;

(i) 4.2×

10<sup>−

10</sup> M epidermal growth factor;

(ii) 2.8×

10<sup>−

10</sup> M basic fibroblast growth factor

(iii) 8.6×

10<sup>−

5</sup> M insulin

(iv) 1.0×

10<sup>−

7</sup> M dexamethasone

(v) 3.2×

10<sup>−

4</sup> M L-ascorbic acid phosphate magnesium salt n-hydrate

(vi) 2×

10<sup>−

10</sup> M L-3,3′

,5-triiodothyronine

(vii) 10<sup>−

4</sup> M ethanolamine

(viii) 3.9×

10<sup>−

8</sup> M selenious acid

(ix) 4×

10<sup>−

3</sup> M Glutamax

(x) 3.3×

10<sup>−

6</sup> M glutathione (reduced); and

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

21. The construct of claim 20, wherein the serum-free defined chemical media further comprises an additional component selected from the group consisting of platelet derived growth factor in a concentration of from 0.1 ng/ml to 10 ng/ml;

L-cysteine in a concentration range of from 0.1 mM to 1 mM, and combinations thereof.

22. The construct of claim 15, wherein the cells are seeded at a density of between 200,000 to 1,000,000 cells/cm².

23. The construct of claim 15, wherein the medium is replaced every 2-3 days.

25. The construct of claim 13, wherein said construct:

(a) is seeded with additional cells that grow and are integrated three dimensionally throughout said construct;

(b) provides mechanical support for said additional cells; and

(c) provides components for development of said additional cells.

38. The construct of claim 13, wherein said construct has a tensile strength of at least 200 kilopascals within three weeks of growth.

41. The construct of claim 13, wherein some of said fibroblast cells are genetically engineered.

42. The construct of claim 13, wherein said cellular migration throughout said construct comprises migration of cells from a subject into said construct upon implantation thereof into said subject.

43. The construct of claim 25, wherein said additional cells, tissue or combination thereof, comprise:

(a) stem cells;

(b) progenitor cells;

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

(d) cells of the 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.

50. The construct of claim 20, wherein the serum-free defined chemical media further comprises an additional component selected from the group consisting of platelet derived growth factor in a concentration of from 0.1 ng/ml to 10 ng/ml;

L-cysteine in a concentration range of from 0.1 mM to 1 mM, and combinations thereof.

14. A three-dimensional cell-produced scaffold construct comprising extracellular matrix synthesized in vitro by fibroblast cells, wherein said construct is produced by a process comprising:

i) incubating the fibroblast cells at a concentration of at least about 200,000 cells/cm² in defined chemical media containing components that promote proliferation and extracellular matrix synthesis of said fibroblast cells; and

ii) isolating the construct from the media,

wherein the construct has a porosity such that it is capable of achieving a thickness of greater than 200 μ

m.

44. The construct of claim 14, wherein the fibroblast cells are seeded at a concentration of at least 200,000 cells/cm² in serum-free defined chemical media.

45. The construct of claim 44, wherein the serum-free defined chemical media comprises base media, basic fibroblast growth factor, epidermal growth factor, dexamethasone, L-3,3′

,5-triiodothyronine, insulin, selenious acid, and a lipid precursor.

46. The construct of claim 45, wherein the base media is selected from the group consisting of Dulbecco'"'"'s Modification of Eagle'"'"'s Medium (DMEM);

Ham'"'"'s F-12 (F12);

Ham'"'"'s F-10 (F10);

Iscove'"'"'s Modification of DMEM (IDMEM);

MEDIUM 199;

Alpha MEM;

Basal Medium Eagle with Earle'"'"'s BSS;

DMEM;

F12 1;

1 with L-Glutamine;

Glasgow'"'"'s MEM with L-glutamine (GMEM);

IMDM with HEPES, with or without L-Glutamine;

L-15, without L-Glutamine;

McCoy'"'"'s 5A Modified Medium;

Medium 199;

MEM Eagle;

MEM Eagle-Earle'"'"'s BSS, with or without L-Glutamine;

MEM Eagle-Hanks BSS;

NCTC-109;

Richter'"'"'s CM Medium;

RPMI 1640 with HEPES;

RPMI 1640 and a combination thereof.

47. The construct of claim 46, wherein the serum-free defined chemical media further comprises additional components selected from the group consisting of platelet derived growth factor (PDGF), L-cysteine, Trolox and combinations thereof.

48. The construct of claim 44, wherein the serum-free defined chemical media comprises:

(i) L-ascorbic acid phosphate magnesium salt n-hydrate in a concentration range of from 0.1 mM to 3 mM;

(ii) epidermal growth factor in a concentration range of from 0.002 nM to 2 nM;

(iii) basic fibroblast growth factor in a concentration range of from 0.03 nM to over 3 nM;

(iv) insulin in a concentration range of from 10 μ

M to 1000 μ

M;

(v) L-3,3′

,5-triiodothyronine in a concentration range of from 0.1 nM to 10 nM;

(vi) ethanolamine in a concentration range of from 1 μ

M to 10,000 μ

M;

(vii) selenious acid in a concentration range of from 10 nM to 1000 nM;

(viii) Glutamax in a concentration range of from 0 mM to 10 mM; and

(ix) dexamethasone in a concentration range of from 1 nM to 1000 nM.

49. The construct of claim 44, wherein the serum-free defined chemical 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 supplemented with the following components;

(i) 4.2×

10<sup>−

10</sup> M epidermal growth factor;

(ii) 2.8×

10<sup>−

10</sup> M basic fibroblast growth factor

(iii) 8.6×

10<sup>−

5</sup> M insulin

(iv) 1.0×

10<sup>−

7</sup> M dexamethasone

(v) 3.2×

10<sup>−

4</sup> M L-ascorbic acid phosphate magnesium salt n-hydrate

(vi) 2×

10<sup>−

10</sup> M L-3,3′

,5-triiodothyronine

(vii) 10<sup>−

4</sup> M ethanolamine

(viii) 3.9×

10<sup>−

8</sup> M selenious acid

(ix) 4×

10<sup>−

3</sup> M Glutamax

(x) 3.3×

10<sup>−

6</sup> M glutathione (reduced); and

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

51. The construct of claim 44, wherein the cells are seeded at a density of between 200,000 to 1,000,000 cells/cm².

52. The construct of claim 44, wherein the medium is replaced every 2-3 days.

53. The construct of claim 14, wherein said construct:

(a) is seeded with additional cells that grow and are integrated three dimensionally throughout said construct;

(b) provides mechanical support for said additional cells; and

(c) provides components for development of said additional cells.