Three dimensional tissues for high-throughput assays

• US 9,631,169 B2
• Filed: 06/05/2009
• Issued: 04/25/2017
• Est. Priority Date: 06/05/2008
• Status: Active Grant

• Current Assignee: InvivoSciences Inc.
• Original Assignee: InvivoSciences Inc.
• Inventors: Wakatsuki, Tetsuro
• Primary Examiner(s): Flinders, Jeremy C
• Application Number:
  US12/996,168
• Publication Number:
  US 20110118143A1
• Time in Patent Office: 2,881 Days
• CPC Class Codes: C12M 23/12 Well or multiwell plates C12M25/04 takes precedence
• CPC Class Codes: C12M 25/14 Scaffolds; Matrices in general C12N5/0068
• CPC Class Codes: C12M 41/46 of cellular or enzymatic activity or functionality, e.g. cell viability
• CPC Class Codes: C12Q 1/02 involving viable microorganisms
• CPC Class Codes: C40B 30/06 by measuring effects on living organisms, tissues or cells
• CPC Class Codes: G01N 33/5082 Supracellular entities, e.g. tissue, organisms

Abstract

Provided herein are methods of detecting responses of bio-artificial tissues to agents by performing assays using three-dimensional bio-artificial tissues. The methods are adaptable to high-throughput platforms.

Claims

1. A method of detecting the response of a tissue to an agent comprising:

(a) contacting a three-dimensional bio-artificial tissue with an agent, the bio-artificial tissue comprising cells and having an extracellular matrix polymerized therearound, the bio-artificial tissue formed, and suspended on a scaffold support, and overlaying the support without a fastener to facilitate tissue adhesion, the scaffold support being positioned above the bottom of a well with a space between the bio-artificial tissue and the bottom of the well,

(b) without removing the bio-artificial tissue from the well, performing a cell-based assay on the bio-artificial tissue in the well, wherein the assay produces an indicator of a physiologic response of the tissue to the agent, and the indicator is suitable for optical detection, and

(c) optically detecting a level of the indicator in the well, wherein the level of the indicator is indicative of the response of the bio-artificial tissue to the agent.

2. The method of claim 1, wherein the cells are selected from the group consisting of a muscle cell, a non-muscle cell, an endothelial cell, and a cardiac cell.

3. The method of claim 1, wherein the well is a well of positioned within a multi-well plate.

4. The method of claim 3, wherein the multi-well plate comprises from 2 to 10,000 wells.

5. The method of claim 1, wherein the scaffold support is a wire frame and the bio-artificial tissue overlays and is attached at a perimeter to a the wire frame.

6. The method of claim 5, wherein the wire is stainless steel wire.

7. The method of claim 1, wherein the cells comprise cells known to be involved in a disease.

8. The method of claim 1, wherein the indicator is a colorimetric or a fluorometric indicator.

9. The method of claim 1, wherein the level of the indicator is detected using a microscope.

10. The method of claim 1, wherein the indicator is a radioactive label.

11. The method of claim 10, wherein the level of the indicator is detected using a scintillator.

12. The method of claim 1, wherein the level of the indicator is detected using a multi-well plate reader.

13. The method of claim 1, wherein the assay is selected from the group consisting of cell proliferation assays, cell death assays, apoptosis assays, protein expression assays, gene expression assays, enzymatic assays, and cell signaling assays.

14. The method of claim 1, wherein the agent is a candidate pharmaceutical.

15. The method of claim 1, wherein the method is a high throughput screening method.

16. The method of claim 1, further comprising performing a mechanical measurement of the contacted bio-artificial tissue to determine a change in a mechanical property.

17. The method of claim 1, wherein the bio-artificial tissue comprises cardiac cells and fibroblasts.

20. The method of claim 1, wherein the scaffold has at least two parallel, spaced apart members, each positioned within the well and disposed above and substantially parallel to the bottom of the well;

and having at least one leg for supporting the scaffold above the bottom of the well.

21. The method of claim 1, wherein the scaffold has at least one elongate member, and is disposed within the well above and substantially parallel to the bottom of the well, and having at least one leg for supporting the scaffold above the bottom of the well.

22. The method of claim 1, wherein the bio-artificial tissue is suspended from the scaffold support with a space between the bio-artificial tissue and the bottom of the well.

18. A method of detecting the response of a tissue to an agent comprising:

(a) contacting a three-dimensional bio-artificial tissue with an agent, the bio-artificial tissue comprising cells embedded in an extracellular matrix without the use of a microcarrier material, the bio-artificial tissue formed on and overlaying a scaffold support without a fastener to facilitate tissue adhesion, the scaffold support being positioned above the bottom of a well, (b) performing a cell-based assay on the bio-artificial tissue, wherein the assay produces an indicator of a physiologic response of the tissue to the agent, and (c) detecting a level of the indicator in the well, wherein the level of the indicator is indicative of the response of the bio-artificial tissue to the agent, and the indicator is suitable for optical detection.

19. The method of claim 18, wherein the bio-artificial tissue is attached at a perimeter to a wire frame, the wire frame coupled to the scaffold support.

23. A method for screening the effects of a pharmaceutical agent on a tissue, comprising:

a) contacting an agent with a plurality of three-dimensional bio-artificial tissues suspended from and overlaying a plurality of scaffolds without a fastener to facilitate tissue adhesion to the scaffold;

each of the plurality of scaffolds positioned within a well of a multi-well plate without any mesh covering the bottom of the well to provide an array of locations, wherein the agent contacting each bio-artificial tissue is the same or different;

b) without removing the bio-artificial tissues from the wells, performing a cell-based assay on each bio-artificial tissue in the well, wherein the assay produces an indicator of a physiologic response of the tissue to the agent, and the indicator is suitable for optical detection, and c) optically detecting a level of the indicator in each well, wherein the level of the indicator is indicative of the response of the bio-artificial tissue to the agent in each well.