Three-dimensional matrix for producing living tissue equivalents

• US 20030166274A1
• Filed: 11/15/2002
• Published: 09/04/2003
• Est. Priority Date: 11/15/2001
• Status: Abandoned Application

• Current Assignee: University of Medicine and Dentistry of New Jersey (State of New Jersey)
• Original Assignee: University of Medicine and Dentistry of New Jersey (State of New Jersey)
• Inventors: Strande, Louise, Schuster, Kevin M., Hewitt, Charles W.
• Application Number:
  US10/295,398
• Publication Number:
  US 20030166274A1
• Time in Patent Office: Days
• US Class Current: 435/366
• 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: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/40 Nucleotides, nucleosides or bases
• CPC Class Codes: C12N 2501/135 Platelet-derived growth factor [PDGF]
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/395 Thyroid hormones
• CPC Class Codes: C12N 2502/094 keratinocytes
• CPC Class Codes: C12N 2502/1323 Adult fibroblasts
• CPC Class Codes: C12N 2533/56 Fibrin; Thrombin
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06
• CPC Class Codes: C12N 5/0698 Skin equivalents

Abstract

The primary aspect of the present invention it directed to a three-dimensional matrix, a living tissue equivalent, a tissue microarray, methods of making thereof, and methods of using thereof. In one embodiment of the invention, the three-dimensional matrix comprises fibroblasts and a fibrin matrix. In a preferred embodiment, the three-dimensional matrix comprises blood plasma, thrombin and fibroblasts. The three-dimensional matrix in accordance with this invention may be used to construct living tissue equivalents, including but not limited to skin, blood vessel, bone, tendon, ligaments, and organ equivalents, among many others. Furthermore, the living tissue equivalents may be used to test the effect of various agents and to study the mechanism of disease and the efficacy of various treatment protocols.

Claims

1. A three-dimensional matrix comprising a fibrin matrix and fibroblasts.

2. The three-dimensional matrix of claim 1 wherein the fibrin matrix comprises fibrinogens and means for polymerizing and cross-linking the fibrinogens.

3. The three-dimensional matrix of claim 1 wherein the fibrin matrix comprises fibrinogens and a thrombin.

4. The three-dimensional matrix of claim 1 wherein the fibrin matrix comprises a blood plasma and a thrombin.

5. The three-dimensional matrix of claim 1 further comprising one factor selected from the group consisting of a platelet, a factor XIII, a clotting factor, a fibronectin, a non-Willebrand factor, and a growth factor.

6. The three-dimensional matrix of claim 5 wherein the growth factor is selected the group consisting of TGF-β

, PDGF, FGF, EGF, VEGF, IL-1, TNF and vitamin C.

7. The three-dimensional matrix of claim 1 further comprising a means for accelerating the formation of the three-dimensional matrix.

8. The three-dimensional matrix of claim 1 further comprising a means for enhancing the characteristics of the three-dimensional matrix.

9. The three-dimensional matrix of claim 1 wherein the fibroblasts produce a component selected from the group consisting of a collagen, a elastic fiber, a reticular fiber, a proteoglycan, and a hyaluronic acid.

10. The three-dimensional matrix of claim 1 wherein the fibroblasts are obtained from a mechanical disaggregation of a tissue, an enzymatic dissociation of a tissue, or fibroblast stem cells.

11. The three-dimensional matrix of claim 9 further comprising a means for stimulating the production of the collagen by the fibroblasts.

12. The three-dimensional matrix of claim 9 further comprising a means for inhibiting the rapid multiplication of the fibroblasts.

13. The three-dimensional matrix of claim 1 wherein the three-dimensional matrix is formed into a desired shape.

14. The three-dimensional matrix of claim 13 wherein the desired shape is selected from a group consisting of the shape of a blood vessel, a bone, a bone marrow, a brain, a cartilage, a connective tissue, an eye, an ear, an endocrine organ, an epithelial tissue, a muscle tissue, a hair, a heart, an intestine, a lung, a liver, a kidney, a mouth, a skin, a nail, a nose, a nerve tissue, a pancreas, a respiratory duct, a stomach, an urinary bladder, a spinal cord, a tongue, a tooth, and a reproductive organ.

15. The three-dimensional matrix of claim 13 wherein the desired shape is achieved through forming the three-dimensional matrix in a mold, wherein the mold has the desired shape.

16. The three-dimensional matrix of claim 13 wherein the desired shape is achieved through impregnating the three-dimensional matrix into a biocompatible scaffolding material, wherein the biocompatible scaffolding material forms the desired shape.

17. The three-dimensional matrix of claim 16 wherein the biocompatible scaffolding material is a biodegradable material or a non-biodegradable material.

18. The three-dimensional matrix of claim 1 wherein the three-dimensional matrix is used to culture cells or provide a scaffold for a living tissue equivalent.

19. A method of producing a three-dimensional matrix comprising mixing fibroblasts, thrombin, and fibrinogens or a blood plasma to form a mixture.

20. The method of claim 19 further comprising adding into the mixture a factor selected from the group consisting of a platelet, a factor XIII, a clotting factor, a fibronectin, a non-Willebrand factor, and a growth factor.

21. The method of claim 20 wherein the growth factor is selected the group consisting of TGF-β

, PDGF, FGF, EGF, VEGF, IL-1, TNF and vitamin C.

22. The method of claim 19 wherein the fibroblasts produce a component selected from the group consisting of a collagen, an elastic fiber, a reticular fiber, a proteoglycan, and a hyaluronic acid.

23. The method of claim 19 wherein the fibroblasts are obtained from a mechanical disaggregation of a tissue, an enzymatic dissociation of a tissue, or fibroblast stem cells.

24. The method of claim 19 wherein the mixture is formed into a desired shape.

25. The method of claim 24 wherein the desired shape is selected from a group consisting of the shape of a blood vessel, a bone, a bone marrow, a brain, a cartilage, a connective tissue, an eye, an ear, an endocrine organ, an epithelial tissue, a muscle tissue, a hair, a heart, an intestine, a lung, a liver, a kidney, a mouth, a skin, a nail, a nose, a nerve tissue, a pancreas, a respiratory duct, a stomach, an urinary bladder, a spinal cord, a tongue, a tooth, and a reproductive organ.

26. The method of claim 24 wherein the desired shape of the mixture is achieved through forming the mixture in a mold, wherein the mold has the desired shape.

27. The method of claim 24 wherein the desired shape of the mixture is achieved through impregnating the mixture into a biocompatible scaffolding material, wherein the biocompatible scaffolding material forms the desired shape.

28. The method of claim 27 wherein the biocompatible scaffolding material is a biodegradable material or a non-biodegradable material.

29. The method of claim 19 wherein the three-dimensional matrix is used to culture cells or provide a scaffold for a living tissue equivalent.

30. A method of producing a three-dimensional matrix having a desired shape, comprising:

obtaining a mold having the desired shape, and

filing the mold with a mixture of fibroblasts, thrombin, and fibrinogens or a blood plasma.

31. The method of claim 30 wherein the desired shape is selected from a group consisting of the shape of a blood vessel, a bone, a bone marrow, a brain, a cartilage, a connective tissue, an eye, an ear, an endocrine organ, an epithelial tissue, a muscle tissue, a hair, a heart, an intestine, a lung, a liver, a kidney, a mouth, a skin, a nail, a nose, a nerve tissue, a pancreas, a respiratory duct, a stomach, an urinary bladder, a spinal cord, a tongue, a tooth, and a reproductive organ.

32. A method of producing a three-dimensional matrix having a desired shape, comprising:

mixing fibroblasts, thrombin, and fibrinogens or a blood plasma irito a mixture;

selecting a biocompatible material;

constructing the biocompatible materials to form the desired shape; and

impregnating the mixture into the biocompatible materials having the desired shape.

33. The method of claim 32 wherein the desired shape is selected from a group consisting of the shape of a blood vessel, a bone, a bone marrow, a brain, a cartilage, a connective tissue, an eye, an ear, an endocrine organ, an epithelial tissue, a muscle tissue, a hair, a heart, an intestine, a lung, a liver, a kidney, a mouth, a skin, a nail, a nose, a nerve tissue, a pancreas, a respiratory duct, a stomach, an urinary bladder, a spinal cord, a tongue, a tooth, and a reproductive organ.

34. The method of claim 32 wherein the biocompatible scaffolding material is a biodegradable material or a non-biodegradable material.

35. A living tissue equivalent comprising a fibrin matrix, fibroblasts, and cells, wherein the cells are not fibroblasts.

36. The living tissue equivalent of claim 35 wherein the fibrin matrix comprises fibrinogens and means for polymerizing and cross-linking the fibrinogens.

37. The living tissue equivalent of claim 35 wherein the fibrin matrix comprises fibrinogens and a thrombin.

38. The living tissue equivalent of claim 35 wherein the fibrin matrix comprises a blood plasma and a thrombin.

39. The living tissue equivalent of claim 35 further comprising a factor selected from the group consisting of a platelet, a factor XIII, a clotting factor, a fibronectin, a non-Willebrand factor, and a growth factor.

40. The living tissue equivalent of claim 39 wherein the growth factor is selected the group consisting of TGF-β

, PDGF, FGF, EGF, VEGF, IL-1, TNF and vitamin C.

41. The living tissue equivalent of claim 35 further comprising a means for accelerating the formation of the living tissue equivalent.

42. The living tissue equivalent of claim 35 further comprising a means for enhancing the characteristics of the living tissue equivalent.

43. The living tissue equivalent of claim 35 wherein the fibroblasts produce a component selected from the group consisting of a collagen, a elastic fiber, a reticular fiber, a proteoglycan, and a hyaluronic acid.

44. The living tissue equivalent of claim 35 wherein the fibroblasts are obtained from a mechanical disaggregation of a tissue, an enzymatic dissociation of a tissue, or fibroblast stem cells.

45. The living tissue equivalent of claim 43 further comprising a means for stimulating the production of the collagen by the fibroblasts.

46. The living tissue equivalent of claim 43 further comprising a means for inhibiting the rapid multiplication of the fibroblasts.

47. The living tissue equivalent of claim 35 wherein the living tissue equivalent is formed into a desired shape.

48. The living tissue equivalent of claim 47 wherein the desired shape is selected from a group consisting of the shape of a blood vessel, a bone, a bone marrow, a brain, a cartilage, a connective tissue, an eye, an ear, an endocrine organ, an epithelial tissue, a muscle tissue, a hair, a heart, an intestine, a lung, a liver, a kidney, a mouth, a skin, a nail, a nose, a nerve tissue, a pancreas, a respiratory duct, a stomach, an urinary bladder, a spinal cord, a tongue, a tooth, and a reproductive organ.

49. The living tissue equivalent of claim 47 wherein the desired shape is achieved through forming the living tissue equivalent in a mold, wherein the mold has the desired shape.

50. The living tissue equivalent of claim 47 wherein the desired shape is achieve through impregnating the living tissue equivalent into a biocompatible scaffolding material, wherein the biocompatible scaffolding material forms the desired shape.

51. The living tissue equivalent of claim 50 wherein the biocompatible scaffolding material is a biodegradable material or a non-biodegradable material.

52. The living tissue equivalent of claim 35 wherein the cells are normal cells or diseased cells.

53. The living tissue equivalent of claim 52 wherein the diseased cell are cancer cells.

54. The living tissue equivalent of claim 35 wherein the cells are stem cells, wherein the stem cell are not fibroblast stem cells.

55. The living tissue equivalent of claim 54 wherein the stem cells are selected from the group consisting of epithelial stem cells, connective stem cells, muscle stem cells and nervous stem cells.

56. The living tissue equivalent of claim 35 wherein the cells are genetically modified through a transfection.

57. The living tissue equivalent of claim 56 wherein the transfection method is selected from the group consisting of a cationic lipid complex, a calcium phosphate coprecipitation, an electroporation, a DEAE-Dextran method, a viral infection, a microinjection, and a biolistic particle delivery.

58. The living tissue equivalent of claim 35 wherein the cells are selected from the group consisting of a blood vessel, a bone, a bone marrow, a brain, a cartilage, a connective tissue, an eye, an ear, an endocrine organ, an epithelial tissue, a muscle tissue, a hair, a heart, an intestine, a lung, a liver, a kidney, a mouth, a skin, a nail, a nose, a nerve tissue, a pancreas, a respiratory duct, a stomach, an urinary bladder, a spinal cord, a tongue, a tooth, and a reproductive organ.

59. A method of producing a living tissue equivalent comprising mixing thrombin, fibroblasts, cells, fibrinogens or a blood plasma, wherein the cells are not fibroblasts.

60. The method of claim 59 further comprising adding a factor selected from the group consisting of a platelet, a factor XIII, a clotting factor, a fibronectin, a non-Willebrand factor, and a growth factor.

61. The method of claim 60 wherein the growth factor is selected the group consisting of TGF-β

, PDGF, FGF, EGF, VEGF, IL-1, TNF and vitamin C.

62. The method of claim 59 wherein the fibroblasts produce a component selected from the group consisting of a collagen, a elastic fiber, a reticular fiber, a proteoglycan, and a hyaluronic acid.

63. The method of claim 59 wherein the fibroblasts are obtained from a mechanical disaggregation of a tissue, an enzymatic dissociation of a tissue, or fibroblast stem cells.

64. The method of claim 59 wherein the mixture is formed into a desired shape.

65. The method of claim 64 wherein the desired shape is selected from a group consisting of the shape of a blood vessel, a bone, a bone marrow, a brain, a cartilage, a connective tissue, an eye, an ear, an endocrine organ, an epithelial tissue, a muscle tissue, a hair, a heart, an intestine, a lung, a liver, a kidney, a mouth, a skin, a nail, a nose, a nerve tissue, a pancreas, a respiratory duct, a stomach, an urinary bladder, a spinal cord, a tongue, a tooth, and a reproductive organ.

66. The method of claim 64 wherein the desired shape of the mixture is achieved through forming the three-dimensional matrix in a mold, wherein the mold has the desired shape.

67. The method of claim 64 wherein the desired shape of the mixture is achieve through impregnating the mixture a biocompatible scaffolding material, wherein the biocompatible scaffolding material forms the desired shape.

68. The method of claim 67 wherein the biocompatible scaffolding material is a biodegradable material or a non-biodegradable material.

69. The method of claim 59 wherein the cells are normal cells or diseased cells.

70. The method of claim 69 wherein the diseased cell are cancer cells.

71. The method of claim 59 wherein the cells are stem cells, wherein the stem cell are not fibroblast stem cells.

72. The method of claim 71 wherein the stem cells are selected from the group consisting of epithelial stem cells, connective stem cells, muscle tissue stem cells and nervous stem cells.

73. The method of claim 59 wherein the cells are genetically modified through a transfection.

74. The method of claim 73 wherein the transfection method is selected from the group consisting of a cationic lipid complex, a calcium phosphate coprecipitation, an electroporation, a DEAE-Dextran method, a viral infection, a microinjection, and a biolistic particle delivery.

75. The method of claim 59 wherein the cells are selected from the group consisting of a blood vessel, a bone, a bone marrow, a brain, a cartilage, a connective tissue, an eye, an ear, an endocrine organ, an epithelial tissue, a muscle tissue, a hair, a heart, an intestine, a lung, a liver, a kidney, a mouth, a skin, a nail, a nose, a nerve tissue, a pancreas, a respiratory duct, a stomach, an urinary bladder, a spinal cord, a tongue, a tooth, and a reproductive organ.

76. A method of producing a living tissue equivalent having a desired shape, comprising:

providing a mold having the desired shape; and

filing the mold with a mixture of cells, fibroblasts, thrombin, and fibrinogens or a blood plasma, wherein the cells are not fibroblasts.

77. The method of claim 76 wherein the desired shape is selected from a group consisting of the shape of a blood vessel, a bone, a bone marrow, a brain, a cartilage, a connective tissue, an eye, an ear, an endocrine organ, an epithelial tissue, a muscle tissue, a hair, a heart, an intestine, a lung, a liver, a kidney, a mouth, a skin, a nail, a nose, a nerve tissue, a pancreas, a respiratory duct, a stomach, an urinary bladder, a spinal cord, a tongue, a tooth, and a reproductive organ.

78. A method of producing a living tissue equivalent having a desired shape, comprising:

mixing cells, fibroblasts, thrombin, and fibrinogens or a blood plasma into a mixture, wherein the cells are not fibroblasts;

selecting a biocompatible material;

forming the biocompatible materials tinto the desired shape; and

impregnating the mixture into the biocompatible materials having the desired shape.

79. The method of claim 78 wherein the desired shape is selected from a group consisting of the shape of a blood vessel, a bone, a bone marrow, a brain, a cartilage, a connective tissue, an eye, an ear, an endocrine organ, an epithelial tissue, a muscle tissue, a hair, a heart, an intestine, a lung, a liver, a kidney, a mouth, a skin, a nail, a nose, a nerve tissue, a pancreas, a respiratory duct, a stomach, an urinary bladder, a spinal cord, a tongue, a tooth, and a reproductive organ.

80. The method of claim 78 wherein the biocompatible scaffolding material is a biodegradable material or a non-biodegradable material.

81. A method of testing the effect of an agent, comprising:

mixing a blood plasma or fibrinogen, thrombin, fibroblasts, and cells to form a living tissue equivalent, wherein the cells are not fibroblasts;

contacting the agent to the living tissue equivalent; and

evaluating the effect of the agent on the living tissue equivalent.

82. A method of testing the efficacy of a therapy treatment on a diseased tissue, comprising:

obtaining cells from the diseased tissue;

mixing a blood plasma or fibrinogens, thrombin, fibroblasts and the cells to form a living tissue equivalent, wherein the cells are not fibroblasts;

exposing the living tissue equivalent to the therapy treatment; and

evaluating the efficacy of the therapy treatment.

83. The method of claim 82 wherein the diseased tissue is a cancer tissue.

84. The method of claim 83 wherein the therapy treatment is a cancer therapy treatment.

85. A method of screening an agent, comprising:

mixing a blood plasma or fibrinogens, thrombin, fibroblasts, and cells to form a living tissue equivalent, wherein the cells are not fibroblasts; and

contacting the agent to the living tissue equivalent.

86. A method of expressing a desired protein in a tissue, comprising:

transfecting cells with genetic materials to become transfected cells, wherein the transfected cells are capable of expressing the desired protein;

mixing a blood plasma or fibrinogens, thrombin, fibroblasts, and the transfected cells to form a living tissue equivalent.

87. A method of producing a skin equivalent, comprising:

mixing a blood plasma or fibrinogen, thrombin, and fibroblasts to form a three-dimensional matrix;

growing the three-dimensional matrix in a growth media; and

plating skin cells on the surface of the three-dimensional matrix.

88. The method of claim 87 wherein the skin cells are keratinocytes.

89. The method of claim 87 wherein the skin cells are plated at a concentration of from 1×

10² cells/cm³ to 1×

10⁸ cells/cm³.

90. A method of producing a blood vessel equivalent, comprising:

mixing a blood plasma or fibrinogens, thrombin, first fibroblasts and smooth muscle tissue cells into a mixture;

casting the mixture around a cylindrical member;

plating second fibroblasts on the outer surface of the mixture; and

lining endothelial cells on the internal surface of the mixture.

91. A method of producing a bone tissue equivalent, comprising:

mixing a blood plasma or fibrinogens, thrombin, fibroblasts, and a demineralized bone powder.

92. A method of producing a bone equivalent, comprising:

mixing blood plasma, thrombin, fibroblasts, and undifferentiated cells, wherein the undifferentiated cells differentiate to osteoblasts.

93. A method of producing a cartilage tissue equivalent, comprising:

mixing blood plasma, thrombin, fibroblasts, and chondrocytes, or chondrocte stem cells.

94. A tissue microarray comprising a multiplicity of tissue equivalent blocks, wherein the tissue equivalent blocks are formed through slicing a living tissue equivalent comprising a mixture of a blood plasma or fibrinogens, thrombin, fibroblasts, and cells, wherein the cells are not fibroblasts.

95. A tissue microarray of claim 94 wherein the living tissue equivalent is embedded in a paraffin.

96. The tissue microarray of claim 94 wherein the living tissue equivalent is frozen.

97. The tissue microarray of claim 94 wherein the tissue equivalent blocks are used to probe a target selected from the group consisting of a DNA, a RNA, a protein, and a chemical agents.

98. The tissue microarray of claim 94 wherein the tissue equivalent blocks are used to identify in the living tissue equivalent blocks a molecule selected from the group consisting of a DNA, a RNA, and a protein.

99. A method of producing a tissue microarray, comprising:

mixing a blood plasma or fibrinogens, thrombin, fibroblasts, and cells into a living tissue equivalent, wherein the cells are not fibroblasts;

preparing the living tissue equivalent in a sliceable form;

slicing the living tissue equivalent into a multiplicity of tissue equivalent blocks; and

arraying the tissue equivalent blocks.

100. The method of claim 99 wherein the sliceable form is a paraffin embedding or a frozen section.

101. The method of claim 99 wherein the tissue equivalent blocks are arrayed manually or using a tissue microarraying instrument.

102. A method of replacing a tissue in a body, comprising:

mixing a blood plasma or fibrinogens, thrombin, fibroblasts, and cells into a living tissue equivalent, wherein the cells are not fibroblasts; and

implementing the living tissue equivalent in the body.