CARDIAC TISSUE CONSTRUCTS AND METHODS OF FABRICATION THEREOF

• US 20150313704A1
• Filed: 12/06/2013
• Published: 11/05/2015
• Est. Priority Date: 12/07/2012
• Status: Active Grant

• Current Assignee: The Governing Council of the University of Toronto (University of Toronto)
• Original Assignee: The Governing Council of the University of Toronto (University of Toronto)
• Inventors: Zandstra, Peter, Thavandiran, Nimalan, Radisic, Milica
• Granted Patent: US 10,034,738 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61F 2/02 Prostheses implantable into the body closure means for urethra or rectum or for artificial body openings therefor A61F2/0004
• CPC Class Codes: A61F 2230/0002 Two-dimensional shapes, e.g. cross-sections
• CPC Class Codes: A61F 2240/002 Designing or making customized prostheses
• CPC Class Codes: A61L 2430/20 for reconstruction of the heart, e.g. heart valves
• CPC Class Codes: A61L 27/24 Collagen
• CPC Class Codes: A61L 27/3886 comprising two or more cell types
• 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 25/00 Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
• CPC Class Codes: C12M 35/04 Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2513/00 3D culture
• CPC Class Codes: C12N 2529/00 Culture process characterised by the use of electromagnetic stimulation
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 5/0657 Cardiomyocytes; Heart cells
• CPC Class Codes: C12N 5/0697 Artificial constructs associating cells of different lineages, e.g. tissue equivalents blood vessels C12N5/0691

Abstract

Methods and devices are provided for the formation of cardiac tissue constructs. In some embodiments, methods are provided for forming cardiac tissue constructs that including cardiomyocytes, non-myocytes, and extracellular matrix, and which exhibit properties associated with healthy cardiac tissue. In some embodiments, microfabrication platforms are provided to support the transmission of dynamic electromechanical forces, such that the cardiac microtissue constructs may be formed mimicking the basic microenvironment found in the heart. The microfabrication platform may include retaining features for stabilizing the position of the microtissue construct during its formation, and the microfabrication platform may include a ramped support configured to produce tissue constructs having a ring geometry. In some embodiments, the microfabrication platform may be configured to for the application of point electrical stimulation, and/or to amplify the transduction of force into a visible displacement.

Claims

1. A microfabrication platform for forming a tissue construct, comprising:

a substrate; and

two or more retaining structures supported by said substrate, wherein said retaining structures are positioned to apply tension to a tissue construct seeded on said substrate during formation of the tissue construct;

wherein at least one retaining structure includes a stabilizing feature for stabilizing the position of the tissue construct during its formation; and

wherein said stabilizing feature is provided at an intermediate location between said substrate and a distal end of said retaining structure.

2. The microfabrication platform according to claim 1 wherein said stabilizing feature is a groove or a protuberance.

3. The microfabrication platform according to claim 1 wherein said stabilizing feature comprises a ramped profile.

4. The microfabrication platform according to claim 3 wherein said ramped profile comprises an upper ramped segment extending outwards in an upwards direction, a lower ramped segment extending outwards in a downwards direction, and minimum location between said upper ramped segment and said lower ramped segment, such that the position of the tissue construct is stabilized between the upper ramped segment and the lower ramped segment.

5. The microfabrication platform according to claim 1 wherein two or more of said retaining structures each include a stabilizing feature.

7. The microfabrication platform according to claim 1 wherein a blocking structure is provided between a portion of said two or more retaining structures, and wherein said blocking structure is configured to prevent the formation of tissue between the two or more retaining structures during the formation of the tissue construct.

8. The microfabrication platform according to claim 1 wherein one or more of said retaining structures is configured as a cantilever, wherein a deflection of said cantilever is associated with an amount of tension in the tissue construct supported by the retaining structures.

9. The microfabrication platform according to claim 8 wherein a distal end of said cantilever extends beyond a position at which the tissue construct is stabilized by said stabilizing feature, such that deflection of said distal end is amplified.

10. The microfabrication platform according to claim 1 wherein said two or more retaining structures are positioned to produce substantially uniaxial tension within the tissue construct during its formation.

11. The microfabrication platform according to claim 1 wherein a surface of each retaining structure is coated with a poloxamer, wherein said poloxamer is suitable for maintaining relative motion between the tissue construct and the retaining structure during formation of the tissue construct.

12. The microfabrication platform according to claim 1 further comprising a pair of electrodes supported by said substrate and having a relative spacing suitable for point stimulation of the tissue construct.

13. The microfabrication platform according to claim 12 wherein said pair of electrodes is positioned adjacent to one of said retaining structures.

14. The microfabrication platform according to claim 12 wherein said pair of electrodes is positioned to function as one of said retaining structures.

15. The microfabrication platform according to claim 1 wherein said retaining structures are adapted such that the tissue construct exhibits a pathology.

16. The microfabrication platform according to claim 1 further comprising a microwell formed in said substrate, wherein said retaining structures are supported by a base of said microwell.

17. The microfabrication platform according to claim 16 comprising a plurality of said microwells.

61. The microfabrication platform according to claim 1 wherein said substrate comprises a ramped support structure on which said two or more retaining structures are supported.

62. The microfabrication platform according to claim 61 wherein said ramped support structure is provided within a microwell.

63. The microfabrication platform according to claim 62 wherein said ramped support structure is integrally formed within said microwell.

64. The microfabrication platform according to claim 61 wherein said ramped support structure comprises a side wall having a conical shape.

65. The microfabrication platform according to claim 61 wherein said ramped support structure is configured to induce upward motion of a tissue construct during a tissue remodeling process.

66. The microfabrication platform according to claim 61 wherein said ramped support structure comprises an upper platform disposed above a base, and wherein said two or more retaining structures are supported by said upper platform.

67. The microfabrication platform according to claim 61 wherein said two or more retaining structures are integrally formed with said ramped support structure.

68. The microfabrication platform according to claim 62 wherein said microwell comprises a side wall, and wherein at least a portion of said side wall is configured to direct liquid into a portion of said microwell surrounding said ramped support structure.

69. The microfabrication platform according to claim 68 wherein said side wall comprises a lower portion and an upper portion, and wherein said upper portion has a curved shape that is configured to direct liquid into said portion of said microwell surrounding said ramped support structure.

6. (canceled)

18-26. -26. (canceled)

27. A method of forming a cardiac tissue construct using a microfabrication platform;

the microfabrication platform comprising;

a substrate; and

two or more retaining structures supported by said substrate, wherein said retaining structures are positioned to apply tension to a tissue construct seeded on said substrate during formation of the tissue construct;

wherein at least one retaining structure includes a stabilizing feature for stabilizing the position of the tissue construct during its formation; and

wherein said stabilizing feature is provided at an intermediate location between said substrate and a distal end of said retaining structure;

the method comprising;

dispensing, onto the substrate, a mixture comprising collagen mastermix, cardiomyocytes, and fibroblasts, such that the at least two retaining structures are surrounded by the mixture; and

incubating the substrate for a time duration suitable for remodeling of the mixture into the cardiac tissue construct, such that the cardiac tissue construct is suspended between the at least two retaining structures under tension;

wherein a ratio of cardiomyocytes to the total number of cells is provided such that the cardiac tissue construct exhibits a conduction velocity characteristic of healthy human heart tissue.

28. The method according to claim 27 wherein the conduction velocity is between approximately 35 cm/s and 60 cm/s.

29. The method according to claim 27 wherein the conduction velocity is between approximately 44 cm/s and 49 cm/s.

30. The method according to claim 27 wherein the total number of cells is between approximately 25,000 to 100,000 cells.

31. The method according to claim 27 wherein at least one retaining structure includes a stabilizing feature for stabilizing the position of the tissue construct during its formation.

32. The method according to claim 27 wherein an electric field is applied, via point stimulation, to the cardiac tissue construct during its formation.

33. The method according to claim 27 wherein the ratio of cardiomyocytes to the total number of cells is between approximately 55% and 95%.

34. The method according to claim 27 wherein the ratio of cardiomyocytes to the total number of cells is between approximately 60% and 90%.

35. The method according to claim 27 wherein the ratio of cardiomyocytes to the total number of cells is between approximately 65% and 85%.

36. The method according to claim 27 wherein the ratio of cardiomyocytes to the total number of cells is between approximately 70% and 80%.

37. The method according to claim 36 wherein said retaining structures are adapted such that the cardiac tissue construct has a cross-section characterized by a radius that is less than the diffusion limitation threshold, such that the cardiac tissue construct is formed without a diffusion barrier.

70. The method according to claim 27 wherein said substrate comprises a ramped support structure on which said two or more two or more retaining structures are supported, wherein said ramped support structure is configured to raise the tissue construct from a base during the remolding process.

38-60. -60. (canceled)