Acellular bioabsorbable tissue regeneration matrices

• US 8,268,361 B2
• Filed: 10/26/2006
• Issued: 09/18/2012
• Est. Priority Date: 10/26/2005
• Status: Active Grant

• Current Assignee: Genesis Technologies Limited
• Original Assignee: Jan-Eric W. Ahlfors
• Inventors: Ahlfors, Jan-Eric W.
• Primary Examiner(s): FORD, ALLISON M
• Application Number:
  US11/588,840
• Publication Number:
  US 20070202189A1
• Time in Patent Office: 2,154 Days
• Field of Search: None
• US Class Current: 424/529
• CPC Class Codes: A61K 35/30 Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
• CPC Class Codes: A61K 35/545 Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
• CPC Class Codes: A61K 38/16 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof enzyme inhibitors A61K38/005
• CPC Class Codes: A61K 38/385 Serum albumin
• CPC Class Codes: A61K 38/40 Transferrins, e.g. lactoferrins, ovotransferrins
• CPC Class Codes: A61K 38/51 Lyases (4)
• CPC Class Codes: A61K 39/395 Antibodies agglutinins A61K38/36 ; as drug carriers A61K47/50; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• CPC Class Codes: A61K 45/00 Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 9/0002 Galenical forms characterised by the drug release technique; Application systems commanded by energy
• CPC Class Codes: A61L 2300/412 Tissue-regenerating or healing or proliferative agents
• CPC Class Codes: A61L 2300/45 Mixtures of two or more drugs, e.g. synergistic mixtures
• CPC Class Codes: A61L 2430/32 for nerve reconstruction
• CPC Class Codes: A61L 2430/38 for reconstruction of the spine, vertebrae or intervertebral discs
• CPC Class Codes: A61L 2430/40 Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
• 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 27/3616 Blood, e.g. platelet-rich plasma
• 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 27/38 containing added animal cells organs or tissue containing native cells A61L27/36
• CPC Class Codes: A61L 27/54 Biologically active materials, e.g. therapeutic substances A61L27/227 takes precedence
• CPC Class Codes: A61L 27/58 Materials at least partially resorbable by the body
• CPC Class Codes: A61P 1/16 : for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 11/00 : Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 17/00 : Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 : for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 21/00 : Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 21/02 : Muscle relaxants, e.g. for tetanus or cramps
• CPC Class Codes: A61P 25/00 : Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/16 : Anti-Parkinson drugs
• CPC Class Codes: A61P 25/28 : for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system

Abstract

The present invention provides methods and compositions useful in the regeneration of damaged, lost and/or degenerated tissue in humans and animals. In certain embodiments, the present invention provides an acellular bioabsorbable tissue regeneration matrix, methods of making such a matrix, and methods of using such a matrix for the regeneration of damaged, lost and/or degenerated tissue. In certain embodiments, methods and compositions of the present invention are useful in the treatment of damaged, lost and/or degenerated nerve tissue.

Claims

1. A method of producing an acellular bioabsorbable tissue regeneration matrix, said method comprising:

(a) providing an isolated blood sample;

(b) removing intact living cells from said blood sample to obtain an acellular fraction;

(c) combining said acellular fraction with a process medium to form a starting material;

(d) incubating said starting material in an incubation chamber for a first period of time to form an acellular bioabsorbable tissue regeneration matrix; and

(e) isolating said acellular bioabsorbable tissue regeneration matrix;

wherein said acellular bioabsorbable tissue regeneration matrix has a regenerative effect towards cells which is not present in the starting material, and/or wherein said acellular bioabsorbable tissue regeneration matrix has significantly greater regenerative effect towards cells compared to the starting material.

2. The method of claim 1, wherein said blood sample comprises a blood fraction.

3. The method of claim 2, wherein said blood fraction comprises a buffy-coat layer.

4. The method of claim 2, wherein said blood fraction comprises a buffy-coat/platelet/plasma layer.

5. The method of claim 1, wherein said blood sample comprises an anticoagulant.

6. The method of claim 1, wherein said blood sample comprises one or more clots, and said method further comprises solubilizing said one or more clots.

7. The method of claim 1, wherein said removing intact cells is effected by freezing, filtration and/or centrifugation.

8. The method of claim 1, wherein said removing comprises passing the tissue sample through a size selection filter.

9. The method of claim 8, wherein said size selection filter excludes particles larger than about 5 μ

m.

10. The method of claim 8, wherein said size selection filter excludes particles larger than about 1.2 μ

m.

11. The method of claim 8, wherein said size selection filter excludes particles larger than about 0.8 μ

m.

12. The method of claim 1, wherein said acellular fraction is in the form of a liquid solution or suspension.

13. The method of claim 1, wherein said incubation chamber is impermeable to solid or liquid matter.

14. The method of claim 13, wherein said incubation is permeable to gases generated by evaporation of liquid inside the incubation chamber.

15. The method of claim 14, wherein said incubation chamber is incubated in a low humidity environment, such that the evaporation rate of liquid inside the incubation chamber is increased.

16. The method of claim 14, wherein said evaporation of liquid results in an increase in osmolarity of the liquid solution or suspension over the first period of time.

17. The method of claim 1, wherein the osmolarity of the acellular fraction is controlled such that the acellular bioabsorbable tissue regeneration matrix formed during said first period of time is in the form of a three-dimensional matrix.

18. The method of claim 1, wherein the osmolarity of the acellular fraction is controlled such that the acellular bioabsorbable tissue regeneration matrix formed during said first period of time is in the form of a suspension.

19. The method of claim 1, wherein said first period of time is at least 5 days.

20. The method of claim 1, wherein said first period of time is at least 12 days.

21. The method of claim 1, wherein said incubating comprises incubating said acellular sample in a low oxygen environment.

22. The method of claim 21, wherein said low oxygen environment comprises an environment with less than about 5% oxygen.

23. The method of claim 21, wherein said low oxygen environment comprises an environment that substantially lacks oxygen.

24. The method of claim 21, wherein said first period of time is at least 3 days.

25. The method of claim 21, wherein said first period of time is at least 7 days.

26. The method of claim 1, further comprising adding process medium to said isolated blood sample and/or said acellular fraction and/or said starting material

(i) during isolation of the blood sample;

(ii) prior to the removing step;

(iii) prior to the combining step;

(iv) during the combining step;

(v) prior to the incubating step;

(vi) during the incubating step;

or

(vii) any combination thereof.

27. The method of claim 26, wherein said process medium comprises a physiological solution.

28. The method of claim 1, further comprising adding one or more therapeutic agents at one or more steps selected from the group consisting of:

(i) to the blood sample during the isolation thereof prior to (a);

(ii) to the blood sample prior to (b);

(iii) to the acellular fraction prior to (c);

(iv) to the acellular fraction during (c);

(v) to the starting material after (c);

(vi) to the starting material during (d);

(vii) to the regeneration matrix after (d);

(viii) to the isolated regeneration matrix after (e); and

(ix) any combination thereof.

29. The method of claim 28, wherein said one or more therapeutic agents is selected from the group consisting of:

(i) proteins,

(ii) peptides,

(iii) drugs,

(iv) cytokines,

(v) extracellular matrix molecules,

(vi) growth factors, and

(vii) any combination thereof.

30. The method of claim 1, further comprising seeding or mixing said acellular bioabsorbable tissue regeneration matrix with cells after formation thereof, wherein said cells are added during or after (e).

31. The method of claim 30, wherein said cells are selected from the group consisting of:

(i) stem cells,

(ii) progenitor cells,

(iii) somatic cells, and

(iv) any combination thereof.

32. The method of claim 30, wherein the cells are selected from the group consisting of:

embryonic stem cells, neural stem cells, neuroprogenitor cells, neurons, glial cells, and combinations thereof.

33. The method of claim 1, further comprising seeding or mixing said acellular bioabsorbable tissue regeneration matrix with cells while it is being produced.

34. The method of claim 33, wherein said cells are selected from the group consisting of:

(i) stem cells,

(ii) progenitor cells,

(iii) somatic cells, and

(iv) any combination thereof.

35. The method of claim 33, wherein the cells are selected from the group consisting of:

embryonic stem cells, neural stem cells, neuroprogenitor cells, neurons, glial cells, and combinations thereof.

36. The method of claim 1, wherein said acellular bioabsorbable tissue regeneration matrix supports, induces and/or controls an activity selected from the group consisting of:

(i) tissue growth,

(ii) cell growth,

(iii) gene upregulation,

(iv) cell inhibition,

(v) cell proliferation,

(vi) cell dedifferentiation,

(viii) cell transdifferentiation,

(ix) neurite outgrowth,

(x) one or more natural wound healing processes, and

(xi) any combination thereof.

73. An acellular bioabsorbable tissue regeneration matrix generated according to the method of any one of claims 2-15, 26-29, 17-36, 7 or, 37-72 wherein said acellular regeneration matrix:

(a) comprises aggregates of spherical structures having approximately 100 nm to 4 microns in diameter,

(b) is self-assembled;

(c) comprises a proteinaceous core having a majority of protein content derived from blood;

(d) lacks substantial metabolic activity;

or

(e) any combination of (a) to (d).

37. A method of producing an acellular bioabsorbable tissue regeneration matrix, said method comprising:

(a) providing an isolated blood sample;

(b) combining the isolated blood sample with a process medium into a first mixture;

(c) removing intact living cells from said first mixture to obtain an acellular starting material;

(d) incubating said starting material for a first period of time to form an acellular bioabsorbable tissue regeneration matrix; and

(e) isolating said acellular bioabsorbable tissue regeneration matrix;

wherein said acellular bioabsorbable tissue regeneration matrix has a regenerative effect towards cells which is not present in the starting material, and/or wherein said acellular bioabsorbable tissue regeneration matrix has significantly greater regenerative effect towards cells compared to the starting material.

38. The method of claim 37, wherein said blood sample comprises a blood fraction.

39. The method of claim 38, wherein said blood fraction comprises a buffy-coat layer.

40. The method of claim 38, wherein said blood fraction comprises a buffy-coat/platelet/plasma layer.

41. The method of claim 37, wherein said blood sample comprises an anticoagulant.

42. The method of claim 37, wherein said blood sample comprises one or more clots, and said method further comprises solubilizing said one or more clots.

43. The method of claim 37, wherein said removing intact cells is effected by freezing, filtration and/or centrifugation.

44. The method of claim 37, wherein said removing comprises passing the first mixture through a size selection filter.

45. The method of claim 44, wherein said size selection filter excludes particles larger than about 5 μ

m.

46. The method of claim 44, wherein said size selection filter excludes particles larger than about 1.2 μ

m.

47. The method of claim 44, wherein said size selection filter excludes particles larger than about 0.8 μ

m.

48. The method of claim 37, wherein said acellular starting material is in the form of a liquid solution or suspension.

49. The method of claim 37, wherein said incubation chamber is impermeable to solid or liquid matter.

50. The method of claim 49, wherein said incubation is permeable to gases generated by evaporation of liquid inside the incubation chamber.

51. The method of claim 50, wherein said incubation chamber is incubated in a low humidity environment, such that the evaporation rate of liquid inside the incubation chamber is increased.

52. The method of claim 50, wherein said evaporation of liquid results in an increase in osmolarity of the liquid solution or suspension over the first period of time.

53. The method of claim 37, wherein the osmolarity of the acellular starting material is controlled such that the acellular bioabsorbable tissue regeneration matrix formed during said first period of time is in the form of a three-dimensional matrix.

54. The method of claim 37, wherein the osmolarity of the acellular starting mixture is controlled such that the acellular bioabsorbable tissue regeneration matrix formed during said first period of time is in the form of a suspension.

55. The method of claim 37, wherein said first period of time is at least 5 days.

56. The method of claim 37, wherein said first period of time is at least 12 days.

57. The method of claim 37, wherein said incubating comprises incubating said acellular starting material in a low oxygen environment.

58. The method of claim 57, wherein said low oxygen environment comprises an environment with less than about 5% oxygen.

59. The method of claim 57, wherein said low oxygen environment comprises an environment that substantially lacks oxygen.

60. The method of claim 57, wherein said first period of time is at least 3 days.

61. The method of claim 57, wherein said first period of time is at least 7 days.

62. The method of claim 37, further comprising adding process medium to said isolated blood sample and/or said first mixture and/or said starting material wherein said process medium is added:

(i) during isolation of the blood sample;

(ii) prior to the combining step;

(iii) during the combining step;

(iv) prior to the removing step;

(v) prior to the incubating step;

(vi) during the incubating step;

or

(vii) any combination thereof.

63. The method of claim 62, wherein said process medium comprises a physiological solution.

64. The method of claim 37, further comprising adding one or more therapeutic agents at one or more steps selected from the group consisting of:

(i) to the blood sample during the isolation thereof prior to (a);

(ii) to the blood sample prior to (b);

(iii) to the first mixture during (b);

(iv) to the first mixture prior to (c);

(v) to the starting material after (c);

(vi) to the starting material during (d);

(vii) to the regeneration matrix after (d);

(viii) to the isolated regeneration matrix after (e); and

(ix) any combination thereof.

65. The method of claim 64, wherein said one or more therapeutic agents is selected from the group consisting of:

(i) proteins,

(ii) peptides,

(iii) drugs,

(iv) cytokines,

(v) extracellular matrix molecules,

(vi) growth factors, and

(vii) any combination thereof.

66. The method of claim 37, further comprising seeding or mixing said acellular bioabsorbable tissue regeneration matrix with cells after formation thereof, wherein said cells are added during or after (e).

67. The method of claim 66, wherein said cells are selected from the group consisting of:

(i) stem cells,

(ii) progenitor cells,

(iii) somatic cells, and

(iv) any combination thereof.

68. The method of claim 66, wherein the cells are selected from the group consisting of:

embryonic stem cells, neural stem cells, neuroprogenitor cells, neurons, glial cells, and combinations thereof.

69. The method of claim 37, further comprising seeding or mixing said acellular bioabsorbable tissue regeneration matrix with cells while it is being produced.

70. The method of claim 69, wherein said cells are selected from the group consisting of:

(i) stem cells,

(ii) progenitor cells,

(iii) somatic cells, and

(iv) any combination thereof.

71. The method of claim 69, wherein the cells are selected from the group consisting of:

embryonic stem cells, neural stem cells, neuroprogenitor cells, neurons, glial cells, and combinations thereof.

72. The method of claim 37, wherein said acellular bioabsorbable tissue regeneration matrix supports, induces and/or controls an activity selected from the group consisting of:

(i) tissue growth,

(ii) cell growth,

(iii) gene upregulation,

(iv) cell inhibition,

(v) cell proliferation,

(vi) cell dedifferentiation,

(viii) cell transdifferentiation,

(ix) neurite outgrowth,

(x) one or more natural wound healing processes, and

(xi) any combination thereof.