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

• US 9,222,932 B2
• Filed: 06/03/2014
• Issued: 12/29/2015
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: Organovo Inc. (Organovo Holdings, Inc.)
• Original Assignee: Organovo Inc. (Organovo Holdings, Inc.)
• Inventors: Shepherd, Benjamin R., Robbins, Justin B., Gorgen, Vivian A., Presnell, Sharon C.
• Primary Examiner(s): Ton, Thaian N
• Assistant Examiner(s): Moloye, Titilayo
• Application Number:
  US14/295,226
• Publication Number:
  US 20140287960A1
• Time in Patent Office: 574 Days
• US Class Current: 1/1
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: C12N 2502/14 hepatocytes
• CPC Class Codes: C12N 2502/28 Vascular endothelial cells
• CPC Class Codes: C12N 2533/30 Synthetic polymers
• CPC Class Codes: C12N 2535/10 Patterned coating
• CPC Class Codes: C12N 5/0656 Adult fibroblasts
• CPC Class Codes: C12N 5/0671 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
• 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 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5088 of vertebrates

Abstract

Engineered, living, three-dimensional liver tissue constructs including: one or more layers, wherein each layer contains one or more liver cell types, the one or more layers cohered to form a living, three-dimensional liver tissue construct free of pre-formed scaffold. Also disclosed are arrays and methods of making the same.

Claims

1. An engineered, living, three-dimensional liver tissue construct, the construct comprising at least one compartment defining a planar geometry, the compartment comprising an interior defined by a border, the interior comprising parenchymal cells, the parenchymal cells comprising hepatocytes or hepatocyte-like cells, the border comprising non-parenchymal cells, the non-parenchymal cells comprising endothelial cells;

the border completely surrounding the interior and in continuous contact with the interior, the cells cohered to form a living, three-dimensional liver tissue construct at least 3 cells thick in its smallest dimension and at least 50 μ

m thick in its smallest dimension;

provided that at least one component of the construct was bioprinted and the construct is substantially free of pre-formed scaffold at the time of use in an assay, implantation into a subject, or for use in extracorporeal support of a subject.

2. The construct of claim 1, further comprising an extrusion compound, the extrusion compound improving the suitability of the cells for bioprinting.

3. The construct of claim 1, wherein the hepatocytes or hepatocyte-like cells are derived from one or more of the following sources:

adult mammalian liver tissue;

fetal mammalian liver tissue;

embryonic stem cells (ESC);

induced pluripotent stem cells (IPSC);

adult stem/progenitor cells derived from the liver; and

adult stem/progenitor cells derived from a tissue other than liver.

4. The construct of claim 1, wherein the non-parenchymal cells further comprise one or more of:

vascular cells, fibroblasts, mesenchymal cells, immune cells, Kupffer cells, biliary epithelial cells, biliary epithelial-like cells, sinusoidal endothelial cells, liver-derived stem/progenitor cells, and non-liver-derived stem/progenitor cells.

5. The construct of claim 1, comprising one or more layers.

6. The construct of claim 5, comprising a plurality of layers, at least one layer compositionally or architecturally distinct from at least one other layer to create a laminar geometry.

7. The construct of claim 1, for use in in vitro assays.

8. The construct of claim 7, for use in one or more of:

drug discovery;

drug testing;

preclinical research;

toxicity testing;

absorption, distribution, metabolism, and excretion testing (ADME);

drug metabolism and pharmacokinetics testing (DMPK);

disease modeling;

infectious disease modeling;

host disease modeling;

three-dimensional biology studies; and

cell-based screening.

9. The construct of claim 1, wherein the construct is non-innervated.

20. The construct of claim 1, wherein at least one component of the construct was bioprinted by extrusion of bio-ink onto a surface.

22. The construct of claim 1, wherein the hepatocytes or hepatocyte-like cells are IPSC-derived hepatocyte-like cells.

24. The construct of claim 1, wherein the wherein the hepatocyte or hepatocyte-like cells are an immortalized cell line.

26. The construct of claim 1, wherein the non-parenchymal cells further comprise stellate cells.

10. An array of engineered living, three-dimensional liver tissue constructs, each construct comprising at least one compartment defining a planar geometry, the compartment comprising an interior defined by a border, the interior comprising parenchymal cells, the parenchymal cells comprising hepatocytes or hepatocyte-like cells, the border comprising non-parenchymal cells, the non-parenchymal cells comprising endothelial cells;

the border completely surrounding the interior and in continuous contact with the interior, the cells cohered to form a living, three-dimensional liver tissue construct at least 3 cells thick in its smallest dimension and at least 50 μ

m thick in its smallest dimension;

provided that at least one component of each construct was bioprinted.

11. The array of claim 10, wherein each construct further comprises an extrusion compound, the extrusion compound improving the suitability of the cells for bioprinting.

12. The array of claim 10, wherein each construct is substantially free of pre-formed scaffold at the time of use in an assay.

13. The array of claim 10, wherein the parenchymal cells are derived from one or more of the following sources:

adult mammalian liver tissue;

fetal mammalian liver tissue;

embryonic stem cells (ESC);

induced pluripotent stem cells (IPSC);

adult stem/progenitor cells derived from the liver; and

adult stem/progenitor cells derived from a tissue other than liver.

14. The array of claim 10, wherein each construct comprises stem/progenitor cells that were exposed to one or more differentiation signals.

15. The array of claim 10, wherein each construct further comprises one or more of the following cell types:

vascular cells, fibroblasts, mesenchymal cells, immune cells, cancer cells, Kupffer cells, biliary cells, sinusoidal endothelial cells, liver-derived stem/progenitor cells, and non-liver-derived stem/progenitor cells.

16. The array of claim 10, wherein each construct comprises one or more layers.

17. The array of claim 16, wherein each construct comprises a plurality of layers, at least one layer compositionally or architecturally distinct from at least one other layer to create a laminar geometry.

18. The array of claim 10, for use in in vitro assays.

19. The array of claim 18, for use in one or more of:

drug discovery;

drug testing;

preclinical research;

toxicity testing;

absorption, distribution, metabolism, and excretion testing (ADME);

drug metabolism and pharmacokinetics testing (DMPK);

disease modeling;

infectious disease modeling;

host disease modeling;

three-dimensional biology studies; and

cell-based screening.

21. The array of claim 10, wherein at least one component of the construct was bioprinted by extrusion of bio-ink onto a surface.

23. The array of claim 10, wherein the hepatocyte or hepatocyte-like cells are IPSC-derived hepatocyte-like cells.

25. The array of claim 10, wherein the wherein the hepatocyte or hepatocyte-like cells are an immortalized cell line.

27. The array of claim 10, wherein the non-parenchymal cells further comprise stellate cells.