SELF-ASSEMBLING MULTICELLULAR BODIES AND METHODS OF PRODUCING A THREE-DIMENSIONAL BIOLOGICAL STRUCTURE USING THE SAME

• US 20140220685A1
• Filed: 04/03/2014
• Published: 08/07/2014
• Est. Priority Date: 06/24/2008
• Status: Active Grant

• Current Assignee: Curators of the University of Missouri (University of Missouri)
• Original Assignee: Curators of the University of Missouri (University of Missouri)
• Inventors: FORGACS, Gabor, MARGA, Francoise Suzanne, NOROTTE, Cyrille
• Granted Patent: US 9,556,415 B2
• Time in Patent Office: Days
• US Class Current: 435/375
• CPC Class Codes: A61L 27/222 Gelatin
• CPC Class Codes: A61L 27/225 Fibrin; Fibrinogen
• CPC Class Codes: A61L 27/3625 Vascular tissue, e.g. heart valves
• CPC Class Codes: A61L 27/3691 characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
• CPC Class Codes: A61L 27/3886 comprising two or more cell types
• 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
• CPC Class Codes: B33Y 80/00 Products made by additive manufacturing
• CPC Class Codes: C12M 21/08 for producing artificial tissue or for ex-vivo cultivation of tissue prostheses A61F2/00, grafts A61L27/00
• CPC Class Codes: C12M 23/10 Petri dish crystallising dishes B01L3/06
• CPC Class Codes: C12M 25/06 Plates; Walls; Drawers; Multilayer plates
• CPC Class Codes: C12M 25/14 Scaffolds; Matrices in general C12N5/0068
• CPC Class Codes: C12M 33/00 Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus chemical or physical laboratory apparatus in general B01L, devices for taking cell samples A61B10/0045, withdrawing or distributing predetermined quantities of fluid B01L99/00
• CPC Class Codes: C12M 35/08 Chemical, biochemical or biological means, e.g. plasma jet, co-culture
• CPC Class Codes: C12N 2506/1307 from adult fibroblasts
• CPC Class Codes: C12N 2506/1338 from smooth muscle cells
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 2533/56 Fibrin; Thrombin
• CPC Class Codes: C12N 2533/76 Agarose, agar-agar
• CPC Class Codes: C12N 5/0062 General methods for three-dimensional culture
• CPC Class Codes: C12N 5/0068 : General culture methods using substrates for specific animal cell type C12N5/06
• CPC Class Codes: C12N 5/0602 : Vertebrate cells
• CPC Class Codes: C12N 5/0691 : Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels

Abstract

Structures and methods for tissue engineering include a multicellular body including a plurality of living cells. A plurality of multicellular bodies can be arranged in a pattern and allowed to fuse to form an engineered tissue. The arrangement can include filler bodies including a biocompatible material that resists migration and ingrowth of cells from the multicellular bodies and that is resistant to adherence of cells to it. Three-dimensional constructs can be assembled by printing or otherwise stacking the multicellular bodies and filler bodies such that there is direct contact between adjoining multicellular bodies, suitably along a contact area that has a substantial length. The direct contact between the multicellular bodies promotes efficient and reliable fusion. The increased contact area between adjoining multicellular bodies also promotes efficient and reliable fusion. Methods of producing multicellular bodies having characteristics that facilitate assembly of the three-dimensional constructs are also provided.

Claims

1. An elongate multicellular body comprising a plurality of living cells and tissue culture medium, wherein the cells are cohered to one another and the multicellular body has a length of at least about 1000 microns, the multicellular body having an average cross-sectional area along its length in the range of about 7,850 square microns to about 360,000 square microns.

4. The multicellular body of any one of claims 1-3 wherein the multicellular body is lumenless.

6. The multicellular body of any one of claims 1-5 wherein the multicellular body has a length of at least about 1 centimeter.

7. The multicellular body of any one of claims 1-6 wherein the multicellular body has a length of at least about 5 centimeters.

8. The multicellular body of any one of claims 1-7 wherein the multicellular body has a length of less than about 30 centimeters.

9. The multicellular body of any one of claims 1-8 wherein the multicellular body has a length in the range of about 5 centimeters to about 30 centimeters.

10. A multicellular body as set forth in any one of claims 1-9 wherein the multicellular body has an average cross-sectional area along its length in the range of about 31,400 square microns to about 250,000 square microns.

11. The multicellular body of any one of claims 1-10 wherein the multicellular body has an average cross-sectional area along its length in the range of about 31,400 square microns to about 90,000 square microns.

12. The multicellular body of any one of claims 1-11 wherein the multicellular body is substantially cylindrical and has a substantially circular cross-section.

13. The multicellular body of any one of claims 1-11 wherein the multicellular body consists essentially of a plurality of living cells of a single cell type.

14. The multicellular body of any one of claims 1-11 wherein the multicellular body includes living cells of a first type and at least about 90 percent of the cells are cells of the first type.

15. The multicellular body of any one of claims 1-12 wherein the plurality of living cells comprises a plurality of living cells of a first cell type and a plurality of living cells of a second cell type, the second cell type being different from the first cell type.

16. The multicellular body of claim 15, wherein the living cells of the first cell type are endothelial cells and the living cells of the second cell type are smooth muscle cells.

17. The multicellular body of claim 15 wherein the living cells of the first type are smooth muscle cells and the living cells of the second type are fibroblasts.

18. The multicellular body of claim 15, wherein the living cells of the first type are endothelial cells and the living cells of the second type are fibroblasts.

19. The multicellular body of any one of claims 1-12 wherein the plurality of living cells comprises a plurality of living cells of a first cell type, a plurality of living cells of a second cell type, and a plurality of living cells of a third cell type, wherein each of the first, second and third cell types are different from the others of the first, second, and third cell types.

20. The multicellular body of claim 19 wherein the living cells of the first cell type are endothelial cells, the living cells of the second cell type are smooth muscle cells, and the living cells of the third cell type are fibroblasts.

21. The multicellular body of any one of claims 1-16 further comprising one or more extracellular matrix components or one or more derivatives of one or more extracellular matrix components.

22. The multicellular body of claim 21 wherein the one or more derivatives of one or more extracellular matrix components comprises gelatin.

23. The multicellular body of claim 21 or 22 wherein the one or more extracellular matrix components comprises fibrinogen or fibrin.

24. The multicellular body of any one of claims 1-23 wherein the cohesion of the cells in the multicellular body is sufficiently strong to allow the multicellular body to retain a three-dimensional shape when the multicellular body is supported by a flat surface, said three dimensional shape including a width and a height, the height being at least about one half of the width.

25. The multicellular body of any one of claims 1-24 wherein the cohesion of the cells in the multicellular body is sufficiently strong to allow the multicellular body to retain a three-dimensional shape when the multicellular body is supported by a flat surface, the three-dimensional shape being configured:

(i) to include a contact surface on the bottom of the multicellular body where the multicellular body contacts said surface; and

(ii) so a two-dimensional projection of the three-dimensional shape of the multicellular body onto the flat surface has an area that is greater area than an area of said contact surface.

26. The multicellular body of any one of claims 1-25 wherein the cohesion of the cells in the multicellular body is sufficiently strong to allow the multicellular body to support the weight of another multicellular body that is substantially identical to the multicellular body when said other multicellular body is on top of the multicellular body.

27. The multicellular body of any one of claims 1-26 wherein the cohesion of the cells in the multicellular body is sufficiently strong to allow the multicellular body to be picked up by an implement.

2. An engineered elongate multicellular body comprising a plurality of living cells wherein the cells are cohered to one another and the multicellular body has a length of at least about 1000 microns, the multicellular body having an average cross-sectional area along its length in the range of about 7,850 square microns to about 360,000 square microns.

3. A non-innervated and non-cartilaginous elongate multicellular body comprising a plurality of living cells wherein the cells are cohered to one another and the multicellular body has a length of at least about 1000 microns, the multicellular body having an average cross-sectional area along its length in the range of about 7,850 square microns to about 360,000 square microns.

5. A lumenless elongate multicellular body comprising a plurality of living cells and tissue culture medium, wherein the cells are cohered to one another and the multicellular body has an aspect ratio that is at least about 2.

28. A method of producing an elongate multicellular body comprising a plurality of living cells, the method comprising:

shaping a cell paste comprising a plurality of living cells into an elongate shape; and

incubating the shaped cell paste in a controlled environment to allow the cells to cohere to one another to form the elongate multicellular body.

30. The method of claim 28 or 29 further comprising producing the cell paste by mixing a plurality of living cells with a tissue culture medium.

31. The method of claim 30 wherein the producing comprises compacting the living cells.

32. The method of claim 31 wherein the compacting comprises centrifuging a cell suspension comprising the plurality of living cells.

33. The method of any one of claims 28-31 wherein the shaping comprises retaining the cell paste in a first shaping device to allow the cells to partially cohere to one another in the first shaping device, transferring the partially cohered cell paste to a second shaping device, and retaining the partially cohered cell paste in the second shaping device to form the multicellular body.

34. The method of claim 33 wherein the first shaping device comprises a capillary pipette and the second shaping device comprises a device that allows nutrients and oxygen to be supplied to the cells wile they are retained in the second shaping device.

35. The method of claim 33 or 34 wherein the second shaping device comprises a mold made of a biocompatible material which resists migration and ingrowth of cells into it and resists adherence of cells to it.

36. The method of claim 35 wherein the biocompatible material is selected from the group consisting of polytetrafluoroethylene (PTFE), stainless steel, hyaluronic acid, agarose, agar, and polyethylene glycol.

37. The method of claim 28 or 29 further comprising producing the cell paste by mixing a plurality of living cells with a solution comprising about 10 to about 30 percent gelatin to form a cell suspension and compacting the cell suspension to form the cell paste.

38. The method of claim 28, 29, or 37 further comprising producing the cell paste by mixing a plurality of living cells with a solution comprising about 10-80 mg/ml fibrinogen to form a cell suspension and compacting the cell suspension to form the cell paste.

39. The method of any one of claims 28-38 wherein the cell paste consists essentially of a plurality of living cells of a single cell type.

40. The method of any one of claims 28-38 wherein the cell paste includes living cells of a first cell type and at least about 90 percent of the cells are cells of the first cell type.

41. The method of any one of claims 28-38 wherein the cell paste comprises a plurality of living cells of a first cell type and a plurality of living cells of a second cell type, the second cell type being different from the first cell type.

42. The method of claim 41 wherein the cell paste comprises a plurality of endothelial cells and a plurality of smooth muscle cells.

43. The method of any one of claims 28-42 wherein shaping the cell paste comprises shaping the cell paste into an elongate shape having an average cross-sectional area along its length in the range of about 7,850 square microns to about 360,000 square microns.

44. The method of any one of claims 28-43 wherein shaping the cell paste comprises shaping the cell paste into an elongate shape having an average cross-sectional area along its length in the range of about 31,400 square microns to about 250,000 square microns.

45. The method of any one of claims 28-44 wherein shaping the cell paste comprises shaping the cell paste into an elongate shape having an average cross-sectional area along its length in the range of about 31,400 square microns to about 90,000 square microns.

46. The method of any one of claims 28-45 wherein shaping the cell paste comprises shaping the cell paste into an elongate shape having a length of at least about 1000 microns.

47. The method of any one of claims claim 28-46 wherein shaping the cell paste comprises shaping the cell paste into an elongate shape having a length of at least about 1 centimeter.

48. The method of any one of claims 28-47 wherein shaping the cell paste comprises shaping the cell paste into an elongate shape having a length of at least about 5 centimeters.

49. The method of any one of claims 28-48 wherein shaping the cell paste comprises shaping the cell paste into an elongate shape having a length of less than about 30 centimeters.

50. The method of any of claims 28-49 wherein shaping the cell paste comprises shaping the cell paste into an elongate shape having a length in the range of about 5 centimeters to about 30 centimeters.

51. The method of any one of claims 28-50 wherein shaping the cell paste comprises shaping the cell paste into a substantially cylindrical shape having a substantially circular cross-section.

52. The method of any one of claims 28-51 wherein shaping the cell paste comprises shaping a cell paste comprising a plurality of living cells and one or more extracellular matrix components or one or more derivatives of one or more extracellular matrix components.

53. The method of claim 52 wherein shaping the cell paste comprises shaping a cell paste comprising a plurality of living cells and gelatin.

54. The method of either claim 52 or 53 wherein shaping the cell paste comprises shaping a cell paste comprising a plurality of living cells and fibrinogen.

55. The method of any one of claims 52-54 wherein the shaping comprises retaining the cell paste in a single shaping device to form the multicellular body.

56. The method of any one of claims 53-55 wherein the incubating comprises incubating the shaped cell paste in a single controlled environment to allow the cells to cohere to one another to form the multicellular body.

29. A method of producing a multicellular body comprising a plurality of living cells, the method comprising:

shaping a cell paste comprising a plurality of living cells in a device that holds the cell paste in a three-dimensional shape; and

incubating the shaped cell paste in a controlled environment while it is held in said three-dimensional shape for a sufficient time to produce a body that has sufficient cohesion to support itself on a flat surface.

57. A three-dimensional structure comprising a plurality of non-innervated elongate multicellular bodies, each multicellular body comprising a plurality of living cells cohered to one another, wherein the multicellular bodies are arranged in a pattern in which each multicellular body contacts at least one other multicellular body and the multicellular bodies are not cohered to one another.

63. The three-dimensional structure of claim 57 or 60 further comprising one or more filler bodies, each filler body comprising a biocompatible material that resists migration and ingrowth of cells from the multicellular bodies into the filler bodies and resists adherence of cells in the multicellular bodies to the filler bodies, wherein the filler bodies are arranged in the pattern with the multicellular bodies so each filler body contacts at least one multicellular body or another filler body.

67. The structure of any one of claims 57-65 wherein the multicellular bodies each have an average cross-sectional area along their longitudinal axis in the range of about 7,850 square microns to about 360,000 square microns.

68. The structure of any one of claims 57-67 wherein each of the multicellular bodies has a length of at least about 1000 microns.

69. The structure of any one of claims 57-68 wherein each of the multicellular bodies has a length of less than about 30 centimeters.

70. The structure of any one of claims 57-69 wherein each multicellular body consists essentially of cells a single type of cell.

71. The structure of any one of claims 57-69 wherein each multicellular body includes living cells of a first type and at least about 90 percent of the cells in each multicellular body are cells of the first cell type.

72. The structure of any one of claims 57-69 wherein one or more of the multicellular bodies comprises a plurality of living cells of a first cell type and a plurality of living cells of a second cell type, the second cell type being different from the first cell type.

73. The structure of claim 72 wherein the living cells of the first cell type are endothelial cells and the living cells of the second cell type are smooth muscle cells.

74. The structure of claim 72 wherein the living cells of the first type are smooth muscle cells and the living cells of the second type are fibroblasts.

75. The structure of claim 72 wherein the living cells of the first type are endothelial cells and the living cells of the second type are fibroblasts.

76. The structure of any one of claims 57-69 wherein one or more of the multicellular bodies comprises a plurality of living cells of a first cell type, a plurality of living cells of a second type, and a plurality of living cells of a third cell type, wherein each of the first, second and third cell types are different from the others of the first, second, and third cell types.

77. The structure of claim 76 wherein the living cells of the first cell type are endothelial cells, the living cells of the second cell type are smooth muscle cells, and the living cells of the third cell type are fibroblasts.

78. The structure of any one of claims 57-69 wherein a majority of the cells in each of one or more of the multicellular bodies are cells of a first cell type and a majority of the cells in each of one or more other multicellular bodies are cells of a second cell type, the second cell type being different from the first cell type.

79. The structure of claim 78 wherein the cells of the first cell type are endothelial cells and the cells of the second cell type are smooth muscle cells.

80. The structure of any one of claims 57-79 wherein the multicellular bodies are substantially uniform in shape.

81. The structure of any one of claims 63-80 wherein the filler bodies are substantially uniform in shape.

82. The structure of any one of claims 63-81 wherein the filler bodies and multicellular bodies are substantially identical in shape.

83. The structure of any one of claims 63-82 wherein the filler bodies are elongate in shape.

84. The structure of any one of claims 63-83 wherein at least some of said plurality of multicellular bodies are arranged to form a tube-like structure and at least one of the filler bodies is inside the tube-like structure.

85. The structure of claim 84 wherein at least some of the multicellular bodies are arranged in a hexagonal configuration surrounding said at least one filler body to form said tube-like structure.

86. The structure of claim 84 or 85 wherein each of the multicellular bodies surrounding said at least one filler body comprise a plurality of endothelial cells and a plurality of smooth muscle cells.

87. The structure of claim 86 wherein each of the multicellular bodies surrounding said at least one filler body further comprise a plurality of fibroblasts.

88. The structure of claim 84 or 85 wherein the multicellular bodies include first and second sets of multicellular bodies, each multicellular body in said first set comprising endothelial cells, each multicellular body in said second set comprising smooth muscle cells, the multicellular bodies of the first set being arranged to form an inner layer substantially surrounding said at least one filler body, the multicellular bodies of the second set being arranged to form a second layer substantially surrounding said at least one filler body and the inner layer formed by the first set of multicellular bodies, wherein a ratio of the number of endothelial cells to the number of non-endothelial cells in the first set is greater than a ratio of the number of endothelial cells to the number of non-endothelial cells in the second set.

89. The structure of claim 88 wherein a ratio of the number of smooth muscle cells to the number cells that are not smooth muscle cells in the second set is greater than a ratio of the number of smooth muscle to the number of cells that are not smooth muscle cells in the first set.

90. The structure of claim 88 or 89 wherein the multicellular bodies further include a third set of multicellular bodies, each multicellular body in said third set comprising fibroblasts, the multicellular bodies of the third set being arranged to form a layer substantially surrounding said at least one filler body, said inner layer formed by the first set of multicellular bodies, and said second layer formed by the second set of multicellular bodies, wherein a ratio of the number of fibroblasts to the number of non-fibroblasts in the third set is greater than a ratio of the number of fibroblasts to the number of non-fibroblasts in the first set or the second set.

91. The structure of claim 84 or 85 wherein the multicellular bodies include first and second sets of multicellular bodies, each multicellular body in said first set comprising a plurality of endothelial cells and a plurality of smooth muscle cells, each multicellular body in said second set comprising fibroblasts, the multicellular bodies of the first set being arranged to form an inner layer substantially surrounding said at least one filler body, the multicellular bodies of the second set being arranged to form a layer substantially surrounding said at least one filler body and the inner layer formed by the first set of multicellular bodies, wherein a ratio of the number of fibroblasts to the number of non-fibroblasts in the second set is greater than a ratio of the number of fibroblasts to the number of non-fibroblasts in the first set.

92. The structure of claim 84 or 85 wherein the multicellular bodies include first and second sets of multicellular bodies, each multicellular body in said first set comprising endothelial cells, each multicellular body in said second set comprising a plurality of smooth muscle cells and a plurality of fibroblasts, the multicellular bodies of the first set being arranged to form an inner layer substantially surrounding said at least one filler body, the multicellular bodies of the second set being arranged to form a layer substantially surrounding the at least one filler body and the inner layer formed by the first set of multicellular bodies, wherein a ratio of the number of endothelial cells to the number of non-endothelial cells in the first set is greater than a ratio of the number of endothelial cells to the number of non-endothelial cells in the second set.

93. The structure of any one of claims 63-83 wherein one or more of the filler bodies are lumen-forming filler bodies, said lumen-forming filler bodies being arranged to prevent ingrowth of cells from the multicellular bodies into first and second elongate spaces, wherein an end of the first elongate space is adjacent a side of the second elongate space, said lumen-forming filler bodies being substantially surrounded by the multicellular bodies so the multicellular bodies can fuse to form a branched tubular structure.

94. The structure of claim 93 wherein each of the multicellular bodies comprises endothelial cells and smooth muscle cells.

95. The structure of claim 93 wherein the multicellular bodies include first and second sets of multicellular bodies, each multicellular body in said first set comprising a majority of endothelial cells, each multicellular body in said second said comprising a majority of smooth muscle cells, the multicellular bodies of the first set being arranged to form an inner layer substantially surrounding the lumen-forming filler bodies, the multicellular bodies of the second set being arranged to form a layer substantially surrounding the lumen-forming filler bodies and the inner layer formed by the first set of multicellular bodies.

96. The structure of any one of claims 93-95 wherein the plurality of multicellular bodies includes first and second sets of multicellular bodies,

the multicellular bodies in the first set having longitudinal axes and being positioned generally alongside one another to surround said first elongate space, the longitudinal axes of the multicellular bodies in said first set being generally aligned with said first elongate space, and

the multicellular bodies in the second set having longitudinal axes and being positioned generally alongside one another to surround said second elongate space, the longitudinal axes of the multicellular bodies in said second set being generally aligned with said second elongate space.

97. The structure of any one of claims 57-83 wherein at least some of the multicellular bodies are arranged to form a sheet.

98. The structure of any one of claims 57-97 wherein the multicellular bodies are substantially cylindrical and have substantially circular cross-sections.

99. The structure of any one of claims 57-98 wherein the multicellular bodies are arranged in a pattern in which at least one of the multicellular bodies contacts another of the multicellular bodies along a contact area having a length that is at least about 1 centimeter.

100. The structure of any one of claims 57-99 wherein the multicellular bodies are arranged in a pattern in which at least one of the multicellular bodies contacts another of the multicellular bodies along a contact area having a length that is in the range of about 1 centimeter to about 30 centimeters.

101. The three-dimensional structure of any one of claims 57-100 wherein at least some of the multicellular bodies have an aspect ratio in the range of about 20 to about 2000.

102. The three-dimensional structure of any one of claims 57-101 wherein the multicellular bodies each have a length in the range of about 1 centimeter to about 30 centimeters.

103. The three-dimensional structure of any one of claims 57-102 wherein the cohesion of the cells in the multicellular bodies is sufficiently strong to allow the multicellular bodies to retain a three-dimensional shape when the multicellular bodies are supported by a flat surface, said three dimensional shape including a width and a height, the height being at least about one half of the width.

104. The three-dimensional structure of any one of claims 57-103 wherein the cohesion of the cells in the multicellular bodies is sufficiently strong to allow the multicellular bodies to retain a three-dimensional shape when the multicellular bodies are supported by a flat surface, the three-dimensional shape being configured:

(i) to include a contact surface on the bottom of each multicellular body where the multicellular body contacts said surface; and

(ii) so a two-dimensional projection of the three-dimensional shape of the multicellular body onto the flat surface has an area that is greater area than an area of said contact surface.

105. The three-dimensional structure of any one of claims 57-104 wherein the cohesion of the cells in each multicellular body is sufficiently strong to allow each multicellular body to support the weight of another multicellular body that is substantially identical to the multicellular body when said other multicellular body is on top of the multicellular body.

106. The three-dimensional structure of any one of claims 57-105 wherein the cohesion of the cells in each multicellular body is sufficiently strong to allow each multicellular body to be picked up by an implement.

58. A three-dimensional structure comprising a plurality of engineered elongate multicellular bodies, each multicellular body comprising a plurality of living cells cohered to one another, wherein the multicellular bodies are arranged in a pattern in which each multicellular body contacts at least one other multicellular body and the multicellular bodies are not cohered to one another.

59. A three-dimensional structure comprising a plurality of elongate multicellular bodies, each multicellular body comprising a plurality of living cells cohered to one another and tissue culture medium, wherein the multicellular bodies are arranged in a pattern in which each multicellular body contacts at least one other multicellular body and the multicellular bodies are not cohered to one another.

60. A three-dimensional structure comprising:

a plurality of non-innervated multicellular bodies, each multicellular body comprising a plurality of living cells cohered to one another;

wherein the multicellular bodies are arranged in a pattern in which at least one of the multicellular bodies contacts another of the multicellular bodies along a contact area having a length that is at least about 1000 microns.

61. A three-dimensional structure comprising:

a plurality of engineered multicellular bodies, each multicellular body comprising a plurality of living cells cohered to one another;

wherein the multicellular bodies are arranged in a pattern in which at least one of the multicellular bodies contacts another of the multicellular bodies along a contact area having a length that is at least about 1000 microns.

62. A three-dimensional structure comprising:

a plurality of multicellular bodies, each multicellular body comprising a plurality of living cells cohered to one another and tissue culture medium;

wherein the multicellular bodies are arranged in a pattern in which at least one of the multicellular bodies contacts another of the multicellular bodies along a contact area having a length that is at least about 1000 microns.

64. A three-dimensional structure comprising:

a plurality of multicellular bodies, each multicellular body comprising a plurality of living cells cohered to one another; and

a plurality of discrete filler bodies, each filler body comprising a biocompatible material that resists migration and ingrowth of cells from the multicellular bodies into the filler bodies and resists adherence of cells in the multicellular bodies to the filler bodies,

wherein the multicellular bodies and filler bodies are arranged in a pattern in which each multicellular body contacts at least one other multicellular body or at least one filler body.

65. A three-dimensional structure comprising:

a plurality of multicellular bodies, each multicellular body comprising a plurality of living cells cohered to one another; and

a plurality of filler bodies, each filler body comprising a biocompatible material that resists migration and ingrowth of cells from the multicellular bodies into the filler bodies and resists adherence of cells in the multicellular bodies to the filler bodies,

wherein the multicellular bodies and the filler bodies are arranged to form a plurality of spaces in the three dimensional structure that are not occupied by the multicellular bodies and that are not occupied by the filler bodies.

66. The structure of claim 65 wherein at least some of the plurality of spaces in the three dimensional structure are filled with tissue culture medium.

107. A method of producing a three-dimensional biological engineered tissue, the method comprising:

arranging a plurality of elongate multicellular bodies according to a pattern such that each of the multicellular bodies contacts at least one other multicellular body, wherein each multicellular body comprises a plurality of living cells,

allowing at least one of the multicellular bodies to fuse with at least one other multicellular body.

108. The method of claim 107 further comprising arranging one or more filler bodies in the pattern with the multicellular bodies so that each filler body contacts at least one multicellular body or another filler body, each filler body comprising a biocompatible material that resists migration and ingrowth of cells from the multicellular bodies into the filler bodies and resists adherence of cells in the multicellular bodies to the filler bodies

110. The method of claim 108 or 109 wherein the arranging comprises arranging the multicellular bodies and the filler bodies to form a three-dimensional structure having a plurality of spaces that are not occupied by the multicellular bodies and that are not occupied by the filler bodies.

111. The method of claim 110 further comprising filling at least some of the plurality of spaces with tissue culture medium.

112. The method of any one of claims 107-111, wherein the arranging results in at least two of the multicellular bodies contacting each other along a contact area having a length that is at least about 1000 microns.

113. The method of any one of claims 108-112, further comprising separating the filler bodies from the fused multicellular bodies to obtain said engineered tissue.

114. The method of any one of claims 108-113, wherein arranging the filler bodies comprises arranging filler bodies which are permeable to nutrients.

115. The method of any one of claims 107-114 wherein arranging the multicellular bodies comprises arranging multicellular bodies which each have an average cross-sectional area along their longitudinal axis in the range of about 7,850 square microns to about 360,000 square microns.

116. The method of any one of claims 107-115 wherein arranging the multicellular bodies comprises arranging multicellular bodies having a length of at least about 1000 microns.

117. The method of any one of claims 107-116 wherein arranging the multicellular bodies comprises arranging multicellular bodies having a length less than about 30 centimeters.

118. The method of any one of claims 107-117 wherein arranging the multicellular bodies comprises arranging multicellular bodies consisting essentially of cells of a single cell type.

119. The method of any one of claims 107-117 wherein arranging the multicellular bodies comprises arranging multicellular bodies wherein each multicellular body includes living cells of a first type and at least about 90 percent of the cells in each multicellular body are cells of the first cell type.

120. The method of any one of claims 107-117 wherein arranging the multicellular bodies comprises arranging one or more multicellular bodies comprising a plurality of living cells of a first cell type and a plurality of living cells of a second cell type, the second cell type being different from the first cell type.

121. The method of claim 120 wherein the living cells of the first type are endothelial cells and the living cells of the second type are smooth muscle cells.

122. The method of claim 120 wherein the living cells of the first type are smooth muscle cells and the living cells of the second type are fibroblasts.

123. The method of claim 120 wherein the living cells of the first type are endothelial cells and the living cells of the second type are fibroblasts.

124. The method of any one of claims 107-117 wherein one or more of the multicellular bodies comprises a plurality of living cells of a first cell type, a plurality of living cells of a second type, and a plurality of living cells of a third cell type, wherein each of the first, second and third cell types are different from the others of the first, second, and third cell types.

125. The method of claim 124 wherein the living cells of the first cell type are endothelial cells, the living cells of the second cell type are smooth muscle cells, and the living cells of the third cell type are fibroblasts.

126. The method of any one of claims 107-121 wherein arranging the multicellular bodies comprises arranging multicellular bodies that are substantially uniform in shape.

127. The method of any one of claims 108-126 wherein arranging the filler bodies comprises arranging filler bodies that are substantially uniform in shape.

128. The method of any one of claims 108-127 wherein arranging the multicellular bodies and filler bodies comprises arranging multicellular bodies and filler bodies that are substantially identical in shape.

129. The method of any one of claims 108-128 wherein arranging the filler bodies comprises arranging filler bodies that are elongate in shape.

130. The method of any one of claims 108-129 wherein arranging the multicellular bodies and the filler bodies comprises arranging at least some of the plurality of multicellular bodies to form a tube-like structure having at least one of the filler bodies inside the tube-like structure.

131. The method of claim 130 wherein arranging the multicellular bodies comprises arranging at least some of the multicellular bodies in a hexagonal configuration surrounding said at least one filler body to form said tube-like structure.

132. The method of any one of claims 130-131 wherein each of the multicellular bodies surrounding said at least one filler body comprise a plurality of endothelial cells and a plurality of smooth muscle cells.

133. The method of claim 132 wherein each of the multicellular bodies surrounding said at least one filler body further comprise a plurality of fibroblasts.

134. The method of any one of claims 130-131 wherein arranging the multicellular bodies comprises arranging a plurality of multicellular bodies including first and second sets of multicellular bodies, each multicellular body in said first set comprising endothelial cells, each multicellular body in said second set comprising smooth muscle cells, the multicellular bodies of the first set being arranged to form an inner layer substantially surrounding said at least one filler body, the multicellular bodies of the second set being arranged to form a second layer substantially surrounding said at least one filler body and the inner layer formed by the first set of multicellular bodies, wherein a ratio of the number of endothelial cells to the number of non-endothelial cells in the first set is greater than a ratio of the number of endothelial cells to the number of non-endothelial cells in the second set.

135. The method of claim 134 wherein a ratio of the number of smooth muscle cells to the number of cells that are not smooth muscle cells in the second set is greater than a ratio of the number of smooth muscle to the number of cells that are not smooth muscle cells in the first set.

136. The method of claim 134 or 135 wherein arranging the multicellular bodies further comprises arranging a plurality of multicellular bodies including a third set of multicellular bodies, each multicellular body in said third set comprising fibroblasts, the multicellular bodies of the third set being arranged to form a layer substantially surrounding said at least one filler body, said inner layer formed by the first set of multicellular bodies, and said second layer formed by the second set of multicellular bodies, wherein a ratio of the number of fibroblasts to the number of non-fibroblasts in the third set is greater than a ratio of the number of fibroblasts to the number of non-fibroblasts in the first set or the second set.

137. The method of claim 130 or 131 wherein arranging the multicellular bodies comprises arranging first and second sets of multicellular bodies, each multicellular body in said first set comprising a plurality of endothelial cells and a plurality of smooth muscle cells, each multicellular body in said second set comprising fibroblasts, the multicellular bodies of the first set being arranged to form an inner layer substantially surrounding said at least one filler body, the multicellular bodies of the second set being arranged to form a layer substantially surrounding said at least one filler body and the inner layer formed by the first set of multicellular bodies, wherein a ratio of the number of fibroblasts to the number of non-fibroblasts in the second set is greater than a ratio of the number of fibroblasts to the number of non-fibroblasts in the first set.

138. The method of claim 130 or 131 wherein arranging the multicellular bodies comprises arranging first and second sets of multicellular bodies, each multicellular body in said first set comprising endothelial cells, each multicellular body in said second set comprising a plurality of smooth muscle cells and a plurality of fibroblasts, the multicellular bodies of the first set being arranged to form an inner layer substantially surrounding said at least one filler body, the multicellular bodies of the second set being arranged to form a layer substantially surrounding the at least one filler body and the inner layer formed by the first set of multicellular bodies, wherein a ratio of the number of endothelial cells to the number of non-endothelial cells in the first set is greater than a ratio of the number of endothelial cells to the number of non-endothelial cells in the second set.

139. The method of any one of claims 130-138 wherein the separating comprises pulling the at least one filler body out of the inside of the tube-like structure.

140. The method of any one of claims 108-129 wherein arranging the filler bodies comprises arranging one or more filler bodies which are lumen-forming filler bodies, said lumen-forming filler bodies being arranged to prevent ingrowth of cells from the multicellular bodies into first and second elongate spaces, wherein an end of the first elongate space is adjacent a side of the second elongate space, said lumen-forming filler bodies being substantially surrounded by the multicellular bodies so the multicellular bodies can fuse to form a branched tubular structure.

141. The method of claim 140 wherein each of the multicellular bodies comprises endothelial cells and smooth muscle cells.

142. The method of claim 140 wherein arranging the multicellular bodies comprises arranging a plurality of multicellular bodies including first and second sets of multicellular bodies, each multicellular body in said first set comprising a majority of endothelial cells, each multicellular body in said second said comprising a majority of smooth muscle cells, the multicellular bodies of the first set being arranged to form an inner layer substantially surrounding the lumen-forming filler bodies, the multicellular bodies of the second set being arranged to form a layer substantially surrounding the lumen-forming filler bodies and the inner layer formed by the first set of multicellular bodies.

143. The method of any one of claims 140-142 wherein arranging the multicellular bodies comprises arranging a plurality of multicellular bodies including first and second sets of multicellular bodies,

the multicellular bodies in the first set having longitudinal axes and being positioned generally alongside one another to surround said first elongate space, the longitudinal axes of the multicellular bodies in said first set being generally aligned with said first elongate space, and

the multicellular bodies in the second set having longitudinal axes and being positioned generally alongside one another to surround said second elongate space, the longitudinal axes of the multicellular bodies in said second set being generally aligned with said second elongate space.

144. The method of any one of claims 107-129 wherein arranging the multicellular bodies comprises arranging at least some of the multicellular bodies to form a sheet.

145. The method of any one of claims 107-144 wherein arranging the multicellular bodies comprises arranging multicellular bodies which are substantially cylindrical and have substantially circular cross-sections.

146. The method of any one of claims 107-145 wherein arranging the multicellular bodies comprises arranging the multicellular bodies in a pattern in which at least one of the multicellular bodies contacts another of the multicellular bodies along a contact area having a length that is at least about 1 centimeter.

147. The method of any one of claims 132-146 wherein arranging the multicellular bodies comprises arranging the multicellular bodies in a pattern in which at least one of the multicellular bodies contacts another of the multicellular bodies along a contact area having a length that is in the range of about 1 centimeter to about 30 centimeters.

148. The method of any one of claims 107-147 wherein arranging the multicellular bodies comprises arranging at least some multicellular bodies having an aspect ratio in the range of about 20 to about 2000.

149. The method of any one of claims 107-148 wherein arranging the multicellular bodies comprises arranging multicellular bodies each having a length in the range of about 1 centimeter to about 30 centimeters.

150. The method of any one of claims 107-149 wherein arranging the multicellular bodies comprises arranging multicellular bodies wherein the cohesion of the cells in the multicellular body is sufficiently strong to allow the multicellular body to retain a three-dimensional shape while supporting itself on a flat surface, said three dimensional shape including a width and a height, the height being at least 50 percent of the width.

151. The method of any one of claims 107-150 wherein arranging the multicellular bodies comprises arranging multicellular bodies wherein the cohesion of the cells in the multicellular body is sufficiently strong to allow the multicellular body to retain a three-dimensional shape when the multicellular body is supported by a flat surface, the three-dimensional shape being configured:

(i) to include a contact surface on the bottom of the multicellular body where the multicellular body contacts said surface; and

(ii) so a two-dimensional projection of the three-dimensional shape of the multicellular body onto the flat surface has an area that is greater area than an area of said contact surface.

152. The method of any one of claims 107-151 wherein arranging the multicellular bodies comprises arranging multicellular bodies wherein the cohesion of the cells in the multicellular body is sufficiently strong to allow the multicellular body to support the weight of another multicellular body that is substantially identical to the multicellular body when said other multicellular body is on top of the multicellular body.

153. The method of any one of claims 107-152 wherein arranging the multicellular bodies comprises arranging multicellular bodies wherein the cohesion of the cells in the multicellular body is sufficiently strong to allow the multicellular body to be picked up by an implement.

154. The method of any one of claims 107-153 wherein the arranging is automated.

109. A method of producing a three-dimensional biological engineered tissue, the method comprising:

arranging a plurality of multicellular bodies and a plurality of filler bodies according to a pattern such that each of the multicellular bodies contacts at least one of (i) another multicellular body or (ii) a filler body, wherein each multicellular body comprises a plurality of living cells, and wherein each filler body comprises a biocompatible material that resists migration and ingrowth of cells from the multicellular bodies into the biocompatible material and resists adherence of cells in the multicellular bodies to the filler bodies, and

allowing at least one of the multicellular bodies to fuse with at least one other multicellular body.

155. A three-dimensional structure comprising at least one filler body and a plurality of living cells which are cohered to one another, the cells forming a tubular structure substantially surrounding said at least one filler body, wherein the filler body comprises a compliant biocompatible material that resists migration and ingrowth of cells into the material and which resists adherence of cells to the material.

156. The structure of claim 155 wherein the biocompatible material is permeable to nutrients.

157. The structure of claim 155 or 156 wherein the tubular structure has a length of at least about 1000 microns.

158. The structure of any one of claims 155-157 wherein the tubular structure has a length of less than about 30 centimeters.

159. The structure of any one of claims 155-158 wherein the tubular structure has a length in the range of about 5 centimeters to about 30 centimeters.

160. The structure of any one of claims 155-159 wherein the plurality of living cells consists essentially of cells of a single cell type.

161. The structure of any one of claims 155-159 wherein the plurality of living cells comprises living cells of a first cell type and at least about 90 percent of the cells are cells of the first cell type.

162. The structure of any one of claims 155-159 wherein the plurality of living cells comprises a plurality of living cells of a first cell type and a plurality of living cells of a second cell type, the second cell type being different from the first cell type.

163. The structure of claim 162 wherein the living cells of the first cell type are endothelial cells and the living cells of the second cell type are smooth muscle cells.

164. The structure of claim 162 wherein the living cells of the first type are smooth muscle cells and the living cells of the second type are fibroblasts.

165. The structure of claim 162 wherein the living cells of the first type are endothelial cells and the living cells of the second type are fibroblasts.

166. The structure of any one of claims 155-159 wherein the plurality of living cells comprises a plurality of living cells of a first cell type, a plurality of living cells of a second type, and a plurality of living cells of a third cell type, wherein each of the first, second and third cell types are different from the others of the first, second, and third cell types.

167. The structure of claim 166 wherein the living cells of the first cell type are endothelial cells, the living cells of the second cell type are smooth muscle cells, and the living cells of the third cell type are fibroblasts.

168. The structure of claim 163 wherein the endothelial cells form an inner layer substantially surrounding said at least one filler body and the smooth muscle cells form a layer substantially surrounding said at least one filler body and the inner layer formed by the endothelial cells.

169. The structure of claim 166 wherein the endothelial cells form an inner layer substantially surrounding said at least one filler body, the smooth muscle cells form a second layer substantially surrounding said at least one filler body and the inner layer formed by the endothelial cells, and the fibroblasts form a third layer substantially surrounding said at least one filler body, the inner layer formed by the endothelial cells, and the second layer formed by the smooth muscle cells.

170. The structure of any one of claims 155-168 wherein the living cells form a branched tubular structure substantially surrounding one or more of the filler bodies which are lumen-forming filler bodies, said lumen-forming filler bodies being arranged to prevent ingrowth of the living cells into first and second elongate spaces, wherein an end of the first elongate space is adjacent a side of the second elongate space.

171. The structure of any one of claims 155-170 wherein the compliant biocompatible material is selected from the group consisting of agarose, agar, hyaluronic acid, and polyethylene glycol.

172. The structure of any one of claims 155-171 wherein the at least one filler body is separable from the tubular structure by pulling the filler body out of the tubular structure.

173. A mold for producing a multicellular body comprising a plurality of living cells cohered to one another, the mold comprising a biocompatible substrate that is resistant to migration and ingrowth of cells into the substrate and resistant to adherence of cells to the substrate, the substrate being shaped to receive a composition comprising plurality of cells having a relatively lower cohesion and hold the composition in a desired shape during a maturation period during which the cohesion increases to form said multicellular body, the desired shape of the multicellular body having a length of at least about 1000 microns and being configured so every cell within the multicellular body is no more than about 250 microns from an exterior of the body.

174. A tool for making a mold that is suitable for producing a plurality of multicellular bodies, wherein each body comprises a plurality of living cells cohered to one another, the tool comprising:

a body having a top and a bottom; and

a plurality of fins extending from the bottom of the body, wherein each of the fins has a width in the range of about 100 microns to about 800 microns for forming grooves in a biocompatible gel substrate configured for forming living cells placed in the grooves into elongate multicellular bodies, wherein the fins have longitudinal axes and at least one of the fins is spaced laterally from the longitudinal axis of another of the fins.