Engineered renal tissues, arrays thereof, and methods of making the same

• US 9,481,868 B2
• Filed: 10/06/2015
• Issued: 11/01/2016
• Est. Priority Date: 10/06/2014
• Status: Active Grant

• Current Assignee: Organovo Inc. (Organovo Holdings, Inc.)
• Original Assignee: Organovo Inc. (Organovo Holdings, Inc.)
• Inventors: Nguyen, Deborah Lynn Greene, King, Shelby Marie, Presnell, Sharon C.
• Primary Examiner(s): Kim, Taeyoon
• Application Number:
  US14/876,659
• Publication Number:
  US 20160097039A1
• Time in Patent Office: 392 Days
• US Class Current: 1/1
• CPC Class Codes: C12N 2502/1323 Adult fibroblasts
• CPC Class Codes: C12N 2502/256 Renal cells
• CPC Class Codes: C12N 2502/28 Vascular endothelial cells
• CPC Class Codes: C12N 5/0684 Cells of the urinary tract or kidneys
• CPC Class Codes: C12N 5/0697 Artificial constructs associating cells of different lineages, e.g. tissue equivalents blood vessels C12N5/0691
• CPC Class Codes: G01N 2500/02 Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 33/5014 for testing toxicity
• CPC Class Codes: G01N 33/5044 involving specific cell types

Abstract

Disclosed are renal tissues and arrays thereof that include a layer of renal interstitial tissue, the renal interstitial tissue comprising renal fibroblasts and endothelial cells; and a layer of renal epithelial tissue, the renal epithelial tissue comprising renal tubular epithelial cells, the renal epithelial tissue in contact with the layer of renal interstitial tissue to form a three-dimensional, engineered, biological renal tissue. Also disclosed are methods of fabricating and using the same.

Claims

1. A three-dimensional, engineered, bioprinted, biological renal tubule model comprising:

a. a layer of renal interstitial tissue, the renal interstitial tissue comprising renal fibroblasts and endothelial cells; and

b. a layer of renal epithelial tissue, the renal epithelial tissue comprising renal tubular epithelial cells, the renal epithelial tissue in contact with the layer of renal interstitial tissue to form the three-dimensional, engineered, biological renal tubule model;

provided that the interstitial tissue comprises an interstitial bio-ink, the epithelial tissue comprises an epithelial bio-ink, and form a three-dimensional, engineered, biological renal tubule model; and

wherein the fibroblasts and endothelial cells are the only cells present in the layer of renal interstitial tissue and are present in a ratio of fibroblasts to endothelial cells at which the renal tubule model is planar six days post-printing.

2. The renal tubule model of claim 1, wherein the layer of renal interstitial tissue possess an apical and basolateral surface.

3. The renal tubule model of claim 2, wherein at least 70% of the layer of renal epithelial tissue is in contact with the apical surface of the layer of renal interstitial tissue.

4. The renal tubule model of claim 3, wherein the layer of renal epithelial tissue in continuous contact with the layer of renal interstitial tissue.

5. The renal tubule model of claim 3, wherein the layer of renal epithelial tissue is substantially a monolayer.

6. The renal tubule model of claim 5, wherein renal tubular epithelial cells are the only cells present in the layer of renal epithelial tissue.

7. The renal tubule model of claim 6, wherein at least 70% of the cells in the layer of renal epithelial tissue form tight junctions with another cell of the renal epithelial tissue layer.

8. The renal tubule model of claim 1, wherein the fibroblasts and endothelial cells are present in the layer of renal interstitial tissue at a ratio of about 50:

50 fibroblasts to endothelial cells.

9. The renal tubule model of claim 1, wherein any of the layer of renal interstitial tissue or layer of renal epithelial tissue is at least 70% living cells by volume.

10. The renal tubule model of claim 1, further comprising a biocompatible membrane with a pore size greater than about 0.4 μ

m.

11. The renal tubule model of claim 1, wherein the renal tubular model is between 50 and 150 μ

m thick.

12. The renal tubule model of claim 1, which is of uniform thickness.

13. A plurality of the renal tubule models of claim 1, configured to form an array.

14. A three-dimensional, engineered, bioprinted, biological renal tubule model comprising:

a. a layer of renal interstitial tissue, the renal interstitial tissue comprising renal fibroblasts and endothelial cells; and

b. a monolayer of renal epithelial tissue, the renal epithelial tissue consisting essentially of renal tubular epithelial cells, wherein at least 70% of the layer of renal epithelial tissue is in contact with the apical surface of the layer of renal interstitial tissue to form the three-dimensional, engineered, biological renal tubule model;

provided that the interstitial tissue comprises an interstitial bio-ink, the epithelial tissue comprises an epithelial bio-ink, and form a three-dimensional, engineered, biological renal tubule model that is between 50 and 150 μ

m thick; and

wherein the fibroblasts and endothelial cells are the only cells present in the layer of renal interstitial tissue and are present in a ratio of fibroblasts to endothelial cells at which the renal tubule model is planar six days post-printing.

15. A method of fabricating a three-dimensional, engineered, biological renal tubule model, the method comprising:

a. preparing a renal interstitial bio-ink, the interstitial bio-ink comprising a plurality of interstitial cell types, the interstitial cell types comprising renal fibroblasts and endothelial cells;

b. preparing a renal epithelial bio-ink, the epithelial bio-ink comprising renal tubular epithelial cells;

c. depositing the renal interstitial bio-ink and the renal epithelial bio-ink such that the renal epithelial bio-ink forms a layer on at least one surface of the layer of renal interstitial bio-ink; and

d. maturing the deposited bio-ink in a cell culture media to allow the cells to cohere to form the three-dimensional, engineered, biological renal tubule model;

wherein the fibroblasts and endothelial cells are the only cells present in the layer of renal interstitial tissue and are present in a ratio of fibroblasts to endothelial cells at which the renal tubule model is planar six days post-depositing.

16. The method of claim 15, wherein depositing the renal interstitial tissue bio-ink forms a renal interstitial tissue layer with an apical and basolateral surface.

17. The method of claim 16, wherein at least 70% of the layer of renal epithelial tissue is deposited in contact with the layer of renal interstitial tissue.

18. The method of claim 17, wherein the layer of renal epithelial tissue is deposited in continuous contact with the layer of renal interstitial tissue.

19. The method of claim 17, wherein the renal epithelial bio-ink is deposited in a monolayer.

20. The method of claim 19, wherein renal tubular epithelial cells are the only cells present in the renal epithelial bio-ink.

21. The method of claim 20, wherein greater than 70% of renal epithelial cells of the renal epithelial layer form tight junctions with other renal epithelial cells.

22. The method of claim 15, wherein the fibroblasts and endothelial cells are present in the layer of renal interstitial tissue at a ratio of about 50:

50 fibroblasts to endothelial cells.

23. The method of claim 15, wherein the renal interstitial bio-ink is at least 70% living cells by volume.

24. The method of claim 15, further comprising depositing the renal interstitial bio-ink onto a biocompatible membrane with a pore size greater than 0.4 μ

m.

25. The method of claim 15, wherein the wherein the renal tubule model is between 50 and 150 μ

m thick.

26. The method of claim 15, wherein the renal tubule model is of a uniform thickness.

27. The method of claim 15, wherein three-dimensional, engineered, biological renal tubule models are deposited to form an array.

28. A method of assessing the renal toxicity of a therapeutic agent, the method comprising:

a. preparing a renal interstitial bio-ink, the interstitial bio-ink comprising a plurality of interstitial cell types, the interstitial cell types comprising renal fibroblasts and endothelial cells;

preparing a renal epithelial bio-ink, the epithelial bio-ink comprising renal tubular epithelial cells;

b. depositing the interstitial bio-ink and the epithelial bio-ink such that the epithelial bio-ink forms a layer on at least one surface of the interstitial bio-ink; and

c. maturing the deposited bio-ink in a cell culture media to allow the cells to cohere to form a three-dimensional, engineered, biological renal tubule model;

d. contacting a therapeutic agent with the construct;

e. measuring viability or functionality of the renal tubular epithelial cells; and

f, assessing the renal toxicity of the therapeutic agent based on the measured viability or functionality of the renal tubular epithelial cells

wherein the fibroblasts and endothelial cells are the only cells present in the layer of renal interstitial tissue and are present in a ratio of fibroblasts to endothelial cells at which the renal tubule model is planar six days post-depositing.