Cardiac tissue constructs and methods of fabrication thereof

• US 10,034,738 B2
• Filed: 12/06/2013
• Issued: 07/31/2018
• 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: Thavandiran, Nimalan, Radisic, Milica, Zandstra, Peter
• Primary Examiner(s): Beisner, William H.
• Assistant Examiner(s): Henkel, Danielle B
• Application Number:
  US14/650,227
• Publication Number:
  US 20150313704A1
• Time in Patent Office: 1,698 Days
• 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 microwell;

a ramped support structure provided within said microwell, said ramped support structure comprising an annular ramped surface and an upper platform, wherein said annular ramped surface extends upwardly from a base of said microwell to said upper platform; and

one or more retaining structures provided on said upper platform of said ramped support structure, wherein said one or more retaining structures are positioned to apply tension to a tissue construct within said microwell during formation of the tissue construct.

2. The microfabrication platform according to claim 1 wherein said one or more retaining structures comprise two retaining structures that are provided at a relative separation suitable for applying tension to the tissue construct within said microwell during formation of the tissue construct.

3. The microfabrication platform according to claim 1 wherein said one or more retaining structures comprise a single retaining structure for retaining a ring-shaped tissue construct.

4. The microfabrication platform according to claim 1 wherein at least one retaining structure includes a stabilizing feature for stabilizing the position of the tissue construct during its formation.

5. The microfabrication platform according to claim 4 wherein said stabilizing feature is provided at an intermediate location between an upper portion of said ramped support structure and a distal end of said retaining structure.

6. The microfabrication platform according to claim 4 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.

7. The microfabrication platform according to claim 6 wherein a distal end of said cantilever extends beyond a position at which the tissue construct is stabilized by the stabilizing feature, such that deflection of said distal end is amplified.

8. The microfabrication platform according to claim 1 wherein said ramped support structure is integrally formed with said microwell.

9. The microfabrication platform according to claim 1 wherein said ramped support structure comprises a side wall having a conical shape.

10. The microfabrication platform according to claim 1 wherein said ramped support structure comprises a curved side wall.

11. The microfabrication platform according to claim 1 wherein said ramped support structure is configured to induce upward motion of a tissue construct formed within said microwell during a tissue remodeling process.

12. The microfabrication platform according to claim 1 wherein a volume of said microwell surrounding said ramped support structure is sufficiently large to contain an amount of unpolymerized matrix that is suitable for forming a tissue construct, without initially covering an upper portion of said ramped support structure with unpolymerized matrix.

13. The microfabrication platform according to claim 1 wherein said ramped support structure comprises an upper platform disposed above said base of said microwell, and wherein said one or more retaining structures are supported by said upper platform.

14. The microfabrication platform according to claim 13 wherein said one or more retaining structures are integrally formed with said ramped support structure.

15. The microfabrication platform according to claim 1 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.

16. The microfabrication platform according to claim 15 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.

17. The microfabrication platform according to claim 1 wherein said one or more retaining structures includes two or more retaining structures, and 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.

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

19. 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.

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

21. A microplate comprising a plurality of microfabrication platforms, wherein said plurality of microfabrication platforms are provided according to claim 1.

28. The microfabrication platform according to claim 1 wherein at least one retaining structure comprises a proximal portion and a distal portion, wherein said distal portion is angled relative to said proximal portion.

29. The microfabrication platform according to claim 4 wherein at least one retaining structure comprises a proximal portion and a distal portion, wherein said distal portion is angled relative to said proximal portion such that said distal portion extends in an upward and outward direction relative to said proximal portion to form said stabilizing feature.

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

the microfabrication platform comprising;

a microwell;

a ramped support structure provided within the microwell, the ramped support structure comprising an annular ramped surface and an upper platform, wherein the annular ramped surface extends upwardly from a base of the microwell to the upper platform; and

one or more retaining structures provided on the ramped support structure, wherein the one or more retaining structures are configured to apply tension to a tissue construct within the microwell during formation of the tissue construct;

the method comprising;

dispensing, into the microwell, a cell-containing pre-polymerized matrix that is configured to form the tissue construct according to a remodeling process, wherein the pre-polymerized matrix is dispensed into a region surrounding the ramped support structure without contacting the one or more retaining structures; and

incubating the microwell for a time duration suitable for remodeling of the pre-polymerized matrix into the tissue construct, such that the tissue construct moves upwards along the ramped support structure during the remodeling process and is retrained in a ring geometry by the retaining structures.

23. The method platform according to claim 22 wherein the microwell comprises a side wall comprising a lower portion and an upper portion, and wherein the upper portion has a curved shape that is configured to direct liquid into said portion of said microwell surrounding said ramped support structure, and wherein the step of dispensing the pre-polymerized matrix into the microwell comprises:

dispensing the pre-polymerized matrix such that the pre-polymerized matrix contacts the upper portion of the side wall, such that the pre-polymerized matrix flows into the region surrounding the ramped support structure without centrifugation.

24. The method according to claim 22 further comprising:

during or after formation of the tissue construct, adding one or more drug candidates to the microwell; and

measuring a response of the tissue construct to the drug candidate.

25. The method according to claim 24 wherein the response is measured via optical mapping.

26. The method according to claim 24 wherein one or more of the 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;

the method further comprising;

detecting the response via measurement of the deflection of one or more of the retaining structures configured as the cantilever.

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

a microwell;

a support structure provided within said microwell, said support structure comprising an annular lateral surface and an upper platform, wherein said lateral surface extends upwardly and inwardly from a base of said microwell to said upper platform; and

two or more retaining structures supported by said support structure, wherein said two or more retaining structures are configured to apply tension to a tissue construct within said microwell during formation of the tissue construct;

wherein said lateral surface has a shape configured to raise the tissue construct from said well base while preventing tissue formation between said retaining structures during a tissue remodeling process.