Tissue engineered cellular sheets, methods of making and use thereof

• US 20030138950A1
• Filed: 12/11/2002
• Published: 07/24/2003
• Est. Priority Date: 12/11/2001
• Status: Active Grant

• Current Assignee: Nightingale, Inc.
• Original Assignee: Cytograft Tissue Engineering, Inc
• Inventors: LʼHeureux, Nicolas, McAllister, Todd N.
• Granted Patent: US 7,504,258 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• 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 31/005 Ingredients of undetermined constitution or reaction products thereof
• CPC Class Codes: A61L 31/16 Biologically active materials, e.g. therapeutic substances A61L31/047 takes precedence
• CPC Class Codes: C12N 5/0062 General methods for three-dimensional culture

Abstract

The disclosure provides methods and composition to build living tissue covered stents and the like. These tissue coated stents provide a barrier against cell migration to the lumen of the vessel. Since the tissue can surround and envelope the stent, foreign body responses to the stent material are reduced and delayed. The tissue coating is also relatively impermeable to transmural flow, so the wrapped stent can act as a bypass vessel. The tissue is also robust enough to act as a stand alone vessel, without requiring the presence of the metallic stent. These stents can be endothelialized to reduce thrombosis. The genetic modifications described in this disclosure allow for functional organs to be built that express agents that are anti-restenotic or anti-thrombogenic. The use of tissue manipulation devices further allows for the assembly of complex tissue engineered organs. The tissue manipulation devices provide a method of anchoring the tissue sheets to the cell culture substrate to promote prolonged maturation and subsequent increased mechanical strength. The tissue manipulation devices also provide a technique to facilitate removal of the sheet from the culture container and subsequent production steps to assemble more complex three dimensional organs from the robust sheet. The tissue manipulation devices also facilitate automated handling of the sheets in the assembly processes.

Claims

1. A genetically engineered living stent generated by a method comprising:

culturing a population of adherent cells in a culture container in the presence of one or more tissue manipulation device(s) under conditions to allow the formation of a tissue sheet comprised of cells and extracellular matrix formed by the cells in contact with the one or more tissue manipulation device(s) whereby the one or more tissue manipulation device(s) anchors the tissue sheet in the culture container and wherein at least one cell of the population of cells is transfected with an exogenous polynucleotide such that the exogenous polynucleotide expresses a product;

removing the tissue sheet from the culture container; and

forming a tubular structure with the tissue sheet thereby forming a genetically engineered living stent.

2. The cellular stent of claim 1, further comprising forming the tubular structure around a biocompatible stent mounted on a temporary support mandrel by layering the tissue one or more times around the biocompatible stent.

3. The genetically engineered living stent of claim 1, wherein the one or more tissue manipulation device(s) is removed prior to, during, or after forming the tubular structure.

4. The genetically engineered living stent of claim 1, wherein the population of cells are substantially homogenous.

5. The genetically engineered living stent of claim 4, wherein the population of cells are fibroblasts.

6. The genetically engineered living stent of claim 1, wherein the population of cells comprise cells selected from the group consisting of fibroblasts, endothelial cells, smooth muscle cells, mesenchymal stem cells, hematopoietic stem cells, circulating stem cells, and any combination thereof.

7. The genetically engineered living stent of claim 1, wherein the one or more tissue manipulation device(s) is substantially non-porous.

8. The genetically engineered living stent of claim 1, wherein the one or more tissue manipulation device(s) is comprised of a non-adherent material.

9. The genetically engineered living stent of claim 1, wherein the one or more tissue manipulation device(s) is comprised of a biocompatible and biodegradable material.

10. The genetically engineered living stent of claim 9, wherein the material is selected from the group consisting of cotton, polylactic acid, polyglycolic acid, a blend of polylactic and polyglycolic acid, cat gut sutures, cellulose, gelatin, dextran, and any combination thereof.

11. The genetically engineered living stent of claim 1, wherein the one or more tissue manipulation device(s) is comprised of a non-biodegradable material.

12. The genetically engineered living stent of claim 11, wherein the non-biodegradable material is selected from the group consisting of a magnetic material, a magnetizable material, a polypropylene, a TEFLON, a steel or a steel alloy, a titanium or a titanium alloy, a polystyrene, a glass, and any combination thereof

13. The genetically engineered living stent of claim 2, wherein the biocompatible stent is made of a biodegradable material.

14. The genetically engineered living stent of claim 2, wherein the biocompatible stent is made of a non-biodegradable material.

15. The genetically engineered living stent of claim 1, wherein the product comprises an anti-thrombotic and/or anti-restenosis agent.

16. The genetically engineered living stent of claim 15, wherein the anti-thrombotic agent and/or anti-restenosis agent is selected from the group consisting of a polypeptide, a protein, a peptide, a peptidomimetic, and a polynucleotide.

17. The genetically engineered living stent of claim 16, wherein the polypeptide is selected from the group consisting of an anti-GM-CSF, an anti-TNF, an anti-IL-1, an anti-IL-2, a nitric oxide synthase, a cyclooxygenase, a tissue plasminogen activator, a statins, a retinoblastoma family gene product, a E2F decoy, a AP-1 decoy, a cyclin-dependent kinase inhibitor, a I kappa B alpha, and any combination of the foregoing.

18. The genetically engineered living stent of claim 1, wherein the product is an agent that increases tissue compliance.

19. The genetically engineered living stent of claim 18, wherein the agent is elastin or an agent that increases elastin production.

20. The genetically engineered living stent of claim 2, further comprising culturing the tissue sheet with the biocompatible stent under conditions such that the tissue sheet matures, adheres to itself, and/or adheres to the biocompatible stent.

21. The genetically engineered living stent of claim 1, wherein the tissue sheet is substantially decellularized to provide extracellular matrix materials provided by the population of cells.

22. The genetically engineered living stent of claim 2, wherein the biocompatible stent further comprises a therapeutic agent.

23. The genetically engineered living stent of claim 20, wherein the tissue sheet is substantially decellularized and/or denatured.

24. The genetically engineered living stent of claim 1, further comprising an anastomotic device on the end or ends of the genetically engineered living stent.

25. The genetically engineered living stent of claim 1, wherein the tubular structure is formed by wrapping the genetically engineered tissue sheet directly onto a temporary support mandrel thereby forming a genetically engineered living stent.

26. The genetically engineered living stent of claim 1, wherein the population of cells are isolated from a human biopsy.

27. A method of forming a genetically engineered living stent, comprising:

culturing a population of adherent cells in a culture container in the presence of one or more tissue manipulation device(s) under conditions to allow the formation of a tissue sheet comprised of cells and extracellular matrix formed by the cells in contact with the one or more tissue manipulation device(s) whereby the one or more tissue manipulation device(s) anchors the tissue sheet in the culture container and wherein at least one cell of the population of cells is transfected with an exogenous polynucleotide such that the exogenous polynucleotide expresses a product;

removing the tissue sheet from the culture container; and

forming a tubular structure with the tissue sheet thereby forming a genetically engineered living stent.

28. The method of claim 27, further comprising conditioning the genetically engineered in a bioreactor the subjects the genetically engineered stent to physiological conditions of fluid, flow, and/or pressure.

29. A genetically engineered living stent formed by the method of claim 27.

30. A genetically engineered tissue sheet prepared in vitro, comprising

a population of adherent cells wherein at least one cell of the population of cells is transfected with an exogenous polynucleotide such that the exogenous polynucleotide expresses a product, and

culturing the population of adherent cells in a culture container in the presence of one or more tissue manipulation device(s) such that the population of adherent cells grow to form a tissue sheet in contact with the one or more tissue manipulation device(s) whereby the tissue sheet is anchored within the culture container by the one or more tissue manipulation device(s).

31. The genetically engineered tissue sheet of claim 30, wherein the population of cells are isolated from a human biopsy.

32. The genetically engineered tissue sheet of claim 30, wherein the population of cells are substantially homogenous.

33. The genetically engineered tissue sheet of claim 32, wherein the population of cells are fibroblasts.

34. The genetically engineered tissue sheet of claim 30, wherein the population of cells are selected from the group consisting of fibroblasts, endothelial cells, smooth muscle cells, mesenchymal stem cells, hematopoietic stem cells, circulating stem cells, and any combination thereof.

35. The genetically engineered tissue sheet of claim 30, wherein the one or more tissue manipulation device(s) is substantially non-porous.

36. The genetically engineered tissue sheet of claim 30, wherein the one or more tissue manipulation device(s) is comprised of a material that is substantially non-adherent to cells.

37. The genetically engineered tissue sheet of claim 30, wherein the one or more tissue manipulation device(s) is comprised of a biocompatible and biodegradable material.

38. The genetically engineered tissue sheet of claim 37, wherein the material is selected from the group consisting of cotton, polylactic acid, polyglycolic acid, a blend of polylactic and polyglycolic acid, cat gut sutures, cellulose, gelatin, dextran, and any combination thereof.

39. The genetically engineered tissue sheet of claim 30, wherein the one or more tissue manipulation device(s) is comprised of a non-biodegradable material.

40. The genetically engineered tissue sheet of claim 39, wherein the nonbiodegradable material is selected from the group consisting of a magnetic material, a magnetizable material, a polypropylene, a TEFLON, a steel or a steel alloy, a titanium or a titanium alloy, a polystyrene, a glass, and any combination thereof.

41. A genetically engineered tissue sheet of claim 30, wherein the genetically engineered tissue sheet is removed from the culture container.

42. A method for manipulation of a genetically engineered tissue sheet prepared in vitro, comprising:

culturing a population of adherent cells in a culture container in the presence of one or more tissue manipulation device(s) under conditions to allow the formation of a tissue sheet comprised of cells and extracellular matrix formed by the cells in contact with the one or more tissue manipulation device(s) whereby the one or more tissue manipulation device(s) anchors the tissue sheet in the culture container and wherein at least one cell of the population of cells is transfected with an exogenous polynucleotide such that the exogenous polynucleotide expresses a product;

removing the tissue sheet from the culture container; and

forming a desired tissue structure.

43. The method of claim 42, wherein the desired tissue structure is formed by manipulating the tissue sheet comprising the one or more tissue manipulation device(s) onto a scaffold defining a desired tissue structure.

44. A living stent generated by a method comprising:

culturing a population of adherent cells in a culture container in the presence of one or more tissue manipulation device(s) under conditions to allow the formation of a tissue sheet comprised of cells and extracellular matrix formed by the cells in contact with the one or more tissue manipulation device(s) whereby the one or more tissue manipulation device(s) anchors the tissue sheet in the culture container;

removing the tissue sheet from the culture container; and

contacting a biocompatible tubular structure with the tissue sheet to form one or more layers of the tissue sheet in contact with the tubular structure thereby fonning a living stent.

45. The living stent of claim 44, wherein the one or more tissue manipulation device(s) is removed prior to, during, or after contacting the tubular structure.

46. The living stent of claim 44, wherein the population of cells are substantially homogenous.

47. The living stent of claim 46, wherein the population of cells are fibroblasts.

48. The living stent of claim 44, wherein the population of cells comprise cells selected from the group consisting fibroblasts, endothelial cells, smooth muscle cells, mesenchymal stem cells, hematopoietic stem cells, circulating stem cells, and any combination thereof.

49. The living stent of claim 44, wherein the tissue manipulation device is substantially non-porous.

50. The living stent of claim 44, wherein the tissue manipulation device is comprised of a non-adherent material.

51. The living stent of claim 44, wherein the one or more tissue manipulation device(s) is comprised of a biocompatible and biodegradable material.

52. The living stent of claim 51, wherein the material is selected from the group consisting of cotton, polylactic acid, polyglycolic acid, a blend of polylactic and polyglycolic acid, cat gut sutures, cellulose, gelatin, dextran, and any combination thereof.

53. The living stent of claim 44, wherein the one or more tissue manipulation device(s) is comprised of a non-biodegradable material.

54. The living stent of claim 53, wherein the non-biodegradable material is selected from the group consisting of a magnetic material, a magnetizable material, a polypropylene, a TEFLON, a steel or a steel alloy, a titanium or a titanium alloy, a polystyrene, a glass, and any combination thereof

55. The living stent of claim 44, wherein the tubular structure is a biocompatible stent made of a biodegradable material.

56. The living stent of claim 44, wherein the tubular structure is a biocompatible stent is made of a non-biodegradable material.

57. The living stent of claim 55 or 56, wherein the stent further comprises an antithrombotic and/or anti-restenosis agent.

58. The living stent of claim 57, wherein the anti-thrombotic agent and/or anti-restenosis agent is selected from the group consisting of a polypeptide, a protein, a peptide, a peptidomimetic, and a polynucleotide.

59. The living stent of claim 44, further comprising culturing the tissue sheet with the tubular structure under conditions such that the tissue sheet matures, adheres to itself, and/or adheres to the tubular structure.

60. The living stent of claim 44, wherein the tissue sheet is substantially decellularized to provide extracellular matrix materials provided by the population of cells before or after contacting the tubular structure.

61. The living stent of claim 44, wherein the population of cells are obtained from a human biopsy.

62. The living stent of claim 44, wherein the contacting with the tubular structure comprises wrapping the tissue sheet one or more times around the stent.

63. The living stent of claim 44, wherein the tubular structure is a support mandrel.

64. The living stent of claim 63, wherein the tissue sheet is wrapped one or more times around the support mandrel.

65. The living stent of claim 64, further comprising removing the support mandrel from the wrapped tissue sheet to form a living stent.

66. The living stent of claim 44, wherein an anastomotic device is on the end or ends of the living stent.

67. A composition, comprising:

a biocompatible stent comprising a therapeutic agent; and

a tissue sheet cultured in vitro substantially enveloping the biocompatible stent, wherein the tissue sheet is derived from a population of adherent cells cultured in a culture container in the presence of one or more tissue manipulation device(s), wherein the population of adherent cells grow to form the tissue sheet whereby the tissue sheet is in contact with the one or more tissue manipulation device(s).

68. A composition, comprising:

a tissue sheet of cells formed into a tubular structure by rolling the tissue sheet upon itself one or more times, wherein the tissue sheet is derived from a population of adherent cells cultured in a culture container in the presence of one or more tissue manipulation device(s), wherein the population of adherent cells grow to form the tissue sheet anchored by the one or more tissue manipulation device(s).

69. A living stent generated by a method comprising:

culturing a population of adherent cells in a culture container under conditions to allow the formation of a tissue sheet comprised of living cells and extracellular matrix formed by the cells;

removing the tissue sheet from the culture container; and

forming a tubular structure with the tissue sheet by wrapping the tissue sheet one or more times around a biocompatible stent which is mounted on a temporary support mandrel, thereby forming a living stent.

70. The living stent of claim 69, further comprising layering a second tissue sheet one or more times around the living stent.

71. The living stent of claim 70, wherein the second tissue sheet is produced with cells of a different type than the one used to produce for the first tissue sheet.

72. The living stent of claim 69, wherein the population of cells comprise cells selected from the group consisting of fibroblasts, endothelial cells, mesothelial, smooth muscle cells, mesenchymal stem cell, hematopoietic stem cells, circulating stem cells, and any combination thereof.

73. The living stent of claim 69, further comprising seeding and culturing a cell population in the lumen of the living stent.

74. The living stent of claim 69, further comprising preconditioning the living stent in vitro with fluid flow and/or pressure.

75. The living stent of claim 69, wherein the tissue sheet is cultured under conditions such that the tissue sheet matures, adheres to itself, and/or adheres to the biocompatible stent.

76. The living stent of claim 69, wherein the tissue sheet is decellularized before or after wrapping the tissue sheet about the biocompatible stent.

77. A method of making a living stent comprising:

culturing a population of adherent cells in a culture container under conditions to allow the formation of a tissue sheet comprised of living cells and extracellular matrix formed by the cells;

removing the tissue sheet from the culture container; and

forming a tubular structure with the tissue sheet by wrapping the tissue sheet one or more times around a biocompatible stent which is mounted on a temporary support mandrel, thereby forming a living stent.

78. A genetically engineered living stent generated by a method comprising:

culturing a population of adherent cells in a culture container under conditions to allow the formation of a tissue sheet comprised of living cells and extracellular matrix formed by the cells, wherein at least one cell of the population of cells is transfected with an exogenous polynucleotide such that the exogenous polynucleotide expresses a product;

removing the tissue sheet from the culture container; and

forming a tubular structure with the tissue sheet by wrapping the tissue sheet one or more times around a biocompatible stent which is mounted on a temporary support mandrel, thereby forming a living stent.