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

• US 9,442,105 B2
• Filed: 03/15/2013
• Issued: 09/13/2016
• 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:
  US13/841,430
• Publication Number:
  US 20140274802A1
• Time in Patent Office: 1,278 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 comprising: 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. In some embodiments, the constructs are characterized by having at least one of: at least one layer comprising a plurality of cell types, the cell types spatially arranged relative to each other to create a planar geometry; and a plurality of layers, at least one layer compositionally or architecturally distinct from at least one other layer to create a laminar geometry. Also disclosed are arrays and methods of making the same. Also disclosed are engineered, living, three-dimensional liver tissue constructs for use in the augmentation or restoration of one or more liver functions, by in vivo delivery of tissue or utilization of tissue in an extracorporeal device.

Claims

1. A method of fabricating a living, three-dimensional liver tissue construct comprising:

a. preparing one or more bio-inks comprising non-parenchymal cells;

b. preparing one or more bio-inks comprising parenchymal cells, the parenchymal cells comprising hepatocytes or hepatocyte-like cells;

c. depositing the bio-inks onto a support to form at least one compartment, the compartment comprising an interior comprising parenchymal cells confined by a border comprising non-parenchymal cells, the border completely surrounding the interior and in continuous contact with the interior; and

d. incubating the deposited bio-inks for a duration of about 1 hour to about 30 days to allow the cells to cohere to form a living, three-dimensional liver tissue construct at least 3 cells thick in its smallest dimension or at least 50 μ

m thick in its smallest dimension.

2. The method of claim 1, wherein the non-parenchymal cells comprise one or more of:

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

3. The method of claim 1, 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.

4. The method of claim 1, wherein at least one component of the construct is bioprinted.

5. The method of claim 4, further comprising an extrusion compound, the extrusion compound improving the suitability of the cells for bioprinting.

6. The method of claim 1, wherein the construct is substantially free of any pre-formed scaffold at the time of use in an assay, implantation into a subject, or for use in extracorporeal support of a subject.

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

8. The method of claim 1, wherein hepatocytes or hepatocyte-like cells comprise ESC-derived hepatocyte-like cells or IPSC-derived hepatocyte-like cells.

9. The method of claim 1, wherein the depositing of the bio-inks is repeated one or more times to create a plurality of layers.

10. The method of claim 1, wherein the parenchymal cells or the non-parenchymal cells comprise stem/progenitor cells.

11. The method of claim 10, further comprising exposing the stem/progenitor cells to one or more differentiation signals.