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

• US 10,400,219 B2
• Filed: 05/26/2016
• Issued: 09/03/2019
• 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): Moloye, Titilayo
• Application Number:
  US15/166,006
• Publication Number:
  US 20160272946A1
• Time in Patent Office: 1,195 Days
• 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 thermoreactive polymers, e.g. PNIPAm, C12N2539/10
• 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 comprising:

(a) at least one layer comprising a plurality of cell types spatially arranged relative to each other in a planar geometry;

or

(b) a plurality of layers, at least one layer compositionally or architecturally distinct from at least one other layer in a laminar geometry, wherein the at least one other layer comprises a plurality of cell types spatially arranged relative to each other in a planar geometry;

wherein the plurality of cell types comprises parenchymal cells in contact with non-parenchymal cells in at least one compartment that comprises an interior comprising the parenchymal cells and a border comprising the non-parenchymal cells,

wherein the parenchymal cells comprise hepatocytes or hepatocyte-like cells and the non-parenchymal cells comprise endothelial cells, and

wherein the construct is substantially free of pre-formed scaffold and adherent confinement material at the time of use in an assay, implantation into a subject, or use in extracorporeal support of a subject.

2. The construct of claim 1, wherein at least one component of the construct was bioprinted.

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

4. The construct of claim 1, wherein the perparenchymal cells are derived from one or more of the following sources:

adult mammalian liver tissue;

fetal mammalian liver tissue;

embryonic stem cells (ESC);

ESC-derived hepatocyte-like cells, induced pluripotent stem cells (IPSC);

IPSC-derived hepatocyte-like cells;

adult stem/progenitor cells derived from the liver; and

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

5. The construct of claim 1, wherein the non-parenchymal cells further comprise one or more of the following cell types:

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

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

7. The construct of claim 6, 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.

8. The construct of claim 1, for use in the augmentation of one or more liver functions in humans.

9. The construct of claim 8, for implantation in a subject at a site of injury, disease, or degeneration.

10. The construct of claim 8, for clinical use in extracorporeal devices designed to augment or restore one or more liver functions.

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

12. An array of engineered living, three-dimensional liver tissue constructs, wherein each construct comprises:

(a) at least one layer comprising a plurality of cell types spatially arranged relative to each other in a planar geometry;

or

(b) a plurality of layers, at least one layer compositionally or architecturally distinct from at least one other layer in a laminar geometry, wherein the at least one other layer comprises a plurality of cell types spatially arranged relative to each other in a planar geometry;

wherein the plurality of cell types comprises parenchymal cells in contact with non-parenchymal cells in at least one compartment that comprises an interior comprising the parenchymal cells and a border comprising the non-parenchymal cells,

wherein the parenchymal cells comprise hepatocytes or hepatocyte-like cells and the non-parenchymal cells comprise endothelial cells, and

wherein each construct is substantially free of pre-formed scaffold and adherent confinement material at the time of use in an assay.

13. The array of claim 12, wherein at least one component of each construct was bioprinted.

14. The array of claim 13, wherein each construct further comprises an extrusion compound, the extrusion compound improving the suitability of the cells for bioprinting.

15. The array of claim 12, wherein the perparenchymal cells are derived from one or more of the following sources:

adult mammalian liver tissue;

fetal mammalian liver tissue;

embryonic stem cells (ESC);

ESC-derived hepatocyte-like cells, induced pluripotent stem cells (IPSC);

IPSC-derived hepatocyte-like cells;

adult stem/progenitor cells derived from the liver; and

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

16. The array of claim 12, wherein each construct comprises stem/progenitor cells that were exposed to one or more differentiation signals.

17. The array of claim 16, wherein the differentiation signal comprises one or more of:

biomechanical signals, soluble signals, and physical signals.

18. The array of claim 12, wherein the non-parenchymal cells further comprise one or more of the following cell types:

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

19. The array of claim 12, for use in in vitro assays.

20. The array of claim 19, 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.