Engineered Renal Tissues, Arrays Thereof, and Methods of Making The Same

• US 20170131264A1
• Filed: 09/08/2016
• Published: 05/11/2017
• 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.
• Granted Patent: US 10,094,821 B2
• Time in Patent Office: Days
• 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

1-30. -30. (canceled)

31. 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; and

(c) a layer of basement membrane between the layer of renal interstitial tissue and layer of renal epithelial 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

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

32. The renal tubule model of claim 31, wherein the layer of renal interstitial tissue possess an apical and basolateral surface.

33. The renal tubule model of claim 31, wherein the layer of renal, epithelial tissue is in continuous contact with the layer of basement membrane and wherein the layer of basement membrane is in continuous contact with the layer renal interstitial tissue.

34. The renal tubule model of claim 31, wherein the layer of renal epithelial tissue is substantially a monolayer.

35. The renal tubule model of claim 34, wherein renal tubular epithelial cells are the only cells present in the layer of renal epithelial tissue.

36. The renal tubule model of claim 31, 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.

37. The renal tubule model of claim 31, wherein any of the layer of renal interstitial tissue or layer of renal epithelial tissue is at least 70% living cells by volume.

38. The renal tubule model of claim 31, further comprising a biocompatible membrane with, a pore size greater than about 0.4 μ

m.

39. The renal tubule model, of claim 31, wherein the renal tubular model is between 50 and 150 μ

m thick.

40. The renal tubule model of claim 31, which is of uniform thickness.

41. The renal tubule model of claim 31, wherein the renal tubular epithelial cells are polarized.

42. The renal tubule model of claim 31, wherein the fibroblasts and endothelial cells are the only cells present in the layer of renal interstitial tissue.

43. A plurality of the renal tubule models of claim 31, configured to form an array.

44. 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 polarized renal tubular epithelial cells, 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 are present in a ratio of fibroblasts to endothelial cells at which the renal tubule model is planar six days post-printing.

45. The renal tubule model of claim 44, wherein the fibroblasts and endothelial cells are the only cells present in the layer of renal interstitial tissue.

46. A plurality of the renal tubule models of claim 44, configured to form an array.

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

(a) contacting the therapeutic agent with, a three-dimensional, engineered, bioprinted, biological renal tubule model comprising;

(i) a layer of renal interstitial tissue, the renal interstitial tissue comprising renal fibroblasts and endothelial cells; and

(ii) a layer of renal epithelial tissue, the renal epithelial tissue comprising renal tubular epithelial cells, 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 present in a ratio of fibroblasts to endothelial cells at which the renal tubule model is planar six days post-printing;

(b) measuring viability or functionality of the renal tubular epithelial cells; and

(c) assessing the renal toxicity of the therapeutic agent based on the measured viability or functionality of the renal tubular epithelial cells.

48. The method of claim 47, wherein the model further comprises a layer of basement membrane between the renal interstitial tissue layer and the renal epithelial tissue layer.

49. The method of claim 47, wherein the renal epithelial cells are polarized.

50. The method of claim 47, wherein the fibroblasts and endothelial cells are the only cells present in the layer of renal interstitial tissue.

51. A method of testing the viability of tubular epithelial cells in the presence of a therapeutic agent, comprising

(a) contacting the therapeutic agent with a three-dimensional, engineered, bioprinted, biological renal tubule model comprising:

(i) a layer of renal interstitial tissue, the renal interstitial tissue comprising renal fibroblasts and endothelial cells; and

(ii) a layer of renal epithelial tissue, the renal epithelial tissue comprising renal, tabular epithelial cells, 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 present in a ratio of fibroblasts to endothelial cells at which the renal tubule model is planar six days post-printing;

(b) measuring gamma glutamyl-transferase (GGT) activity in the model compared to a control; and

(c) wherein decreased GGT activity in the model compared to the control is indicative of reduced viability of the renal tubular epithelial cells.

52. The method of claim 51, wherein the model further comprises a layer of basement membrane between the renal interstitial tissue layer and the renal epithelial tissue layer.

53. The method of claim 51, wherein the renal epithelial cells are polarized.

54. The method of claim 51, wherein the fibroblasts and endothelial cells are the only cells present in the layer of renal interstitial tissue.

55. A method of testing the effect of a therapeutic agent a renal transport molecule, comprising

(a) contacting the therapeutic agent with a three-dimensional, engineered, bioprinted, biological renal tubule model comprising:

(i) a layer of renal interstitial tissue, the renal interstitial tissue comprising renal fibroblasts and endothelial cells; and

(ii) a layer of renal epithelial tissue, the renal epithelial tissue comprising renal tubular epithelial cells, 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 present in a ratio of fibroblasts to endothelial cells at which the renal tubule model is planar six days post-printing; and

(b) measuring the transport molecule activity in the model compared to a control.

56. The method of claim 55, wherein the model further comprises a layer of basement membrane between the renal interstitial tissue layer and the renal epithelial tissue layer.

57. The method of claim 55, wherein the renal epithelial cells are polarized.

58. The method of claim 55, wherein the transport molecule activity is excretion and/or uptake of macromolecules.

59. The method of claim 55, wherein the transport molecule activity is albumin transport.

60. The method of claim 55, wherein the fibroblasts and endothelial cells are the only cells present in the layer of renal interstitial tissue.