Engineered Liver Tissues, Arrays Thereof, and Methods of Making the Same

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.

1 .- 62 . (canceled) 
     
     
         63 . A method of fabricating an engineered living, three-dimensional liver tissue construct comprising:
 preparing one or more bio-inks comprising liver cells;   depositing the one or more bio-inks onto a support; and   incubating the one or more deposited bio-inks for a duration of about 1 hour to about 30 days;   wherein the construct comprises one or more layers, each layer comprising one or more cell types, the one or more layers cohered to form a living, three-dimensional liver tissue construct.   
     
     
         64 . The method of  claim 63 , wherein the liver 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. 
     
     
         65 . The method of  claim 63 , further comprising one or more of the following cell types: vascular cells, endothelial cells, parenchymal cells, non-parenchymal 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. 
     
     
         66 . The method of  claim 63 , wherein the tissue construct is 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.   
     
     
         67 . The method of  claim 63 , wherein at least one component of the construct is bioprinted. 
     
     
         68 . The method of  claim 67 , further comprising an extrusion compound, the extrusion compound improving the suitability of the cells for bioprinting. 
     
     
         69 . The method of  claim 63 , wherein the construct is substantially free of any pre-formed scaffold at the time of use. 
     
     
         70 . The method of  claim 63 , wherein the construct is non-innervated. 
     
     
         71 .- 78 . (canceled) 
     
     
         79 . The method of  claim 65 , further comprising exposing the liver-derived stem/progenitor cells, the non-liver-derived stem/progenitor cells, or both to one or more differentiation signals. 
     
     
         80 . The method of  claim 79 , wherein the differentiation signal comprises one or more of:
 a biomechanical signal, a soluble signal, or a physical signal.   
     
     
         81 . The method of  claim 79 , wherein the stem/progenitor cells were exposed to one or more differentiation signals during fabrication of the construct. 
     
     
         82 . The method of  claim 79 , wherein the stem/progenitor cells were exposed to one or more differentiation signals after fabrication of the construct. 
     
     
         83 . The method of  claim 63 , wherein the construct is for use in in vitro assays. 
     
     
         84 . The method of  claim 81 , wherein the construct is for use in one or more of: tissue-tissue interactions; 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. 
     
     
         85 . The method of  claim 63 , wherein the construct is for use in the augmentation of one or more liver functions in humans. 
     
     
         86 . The method of  claim 85 , wherein the construct is for implantation in a subject at a site of injury, disease, or degeneration. 
     
     
         87 . The method of  claim 85 , wherein the construct is for clinical use in extracorporeal devices designed to augment or restore one or more liver functions. 
     
     
         88 . The method of  claim 63 , wherein a plurality of the liver tissue constructs are constructed. 
     
     
         89 . The method of  claim 88 , further comprising spatially arranging the plurality of liver tissue constructs to form an array. 
     
     
         90 . The method of  claim 89 , wherein the array is use for in cell-based screening, wherein the screening is for one or more infectious diseases, liver fibrosis, liver cancer, liver steatosis, one or more metabolic deficiencies, or one or more protein deficiencies.