Engineered tissues for in vitro research uses, arrays thereof, and methods of making the same

Abstract

Disclosed are living, three-dimensional tissue constructs for in vitro scientific and medical research, arrays thereof, and methods of making said tissues and arrays.

What is claimed is: 
     
         1 . A living, three-dimensional tissue construct comprising: at least one adherent cell type, the at least one adherent cell type cohered and fused to form a living, three-dimensional tissue construct, the tissue construct having a multi-layered architecture which is not a vascular tube, the tissue construct for in vitro use, provided that at least one component of the tissue construct was bioprinted. 
     
     
         2 . The tissue construct of  claim 1 , wherein the tissue construct is substantially free of any pre-formed scaffold at the time of bioprinting or at the time of use. 
     
     
         3 . The tissue construct of  claim 1 , wherein the tissue construct comprises at least one layer comprising a plurality of cell types, the cell types spatially arranged relative to each other to create a planar geometry. 
     
     
         4 . The tissue construct of  claim 1 , wherein the tissue 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. 
     
     
         5 . The tissue construct of  claim 1 , further comprising non-adherent cell types. 
     
     
         6 . The tissue construct of  claim 1 , wherein the tissue construct is secured to a biocompatible surface. 
     
     
         7 . The tissue construct of  claim 6 , wherein the biocompatible surface is a porous membrane. 
     
     
         8 . The tissue construct of  claim 6 , wherein the biocompatible surface is coated with one of or more of the following: a biocompatible hydrogel, a protein, a chemical, a peptide, antibodies, or growth factors. 
     
     
         9 . The tissue construct of  claim 6 , wherein the tissue construct is subjected to shear force, caused by fluid flow, on one or more sides. 
     
     
         10 . The tissue construct of  claim 1 , wherein the tissue construct is at least about 25 μm in its smallest dimension at the time of bioprinting. 
     
     
         11 . The tissue construct of  claim 10 , wherein the tissue construct is no greater than about 3 cm in its largest dimension at the time of bioprinting. 
     
     
         12 . The tissue construct of  claim 1 , for use in in vitro assays. 
     
     
         13 . The tissue construct of  claim 12 , for use in drug testing. 
     
     
         14 . The tissue construct of  claim 1 , wherein the adherent cells are differentiated cells. 
     
     
         15 . The tissue construct of  claim 1 , wherein the adherent cells are non-differentiated cells. 
     
     
         16 . The tissue construct of  claim 1 , wherein the adherent cells originated from a tissue selected from the group consisting of: liver, gastrointestinal, pancreatic, kidney, lung, tracheal, vascular, skeletal muscle, cardiac, skin, smooth muscle, connective tissue, corneal, genitourinary, breast, reproductive, endothelial, epithelial, fibroblast, neural, Schwann, adipose, bone, bone marrow, cartilage, pericytes, mesothelial, endocrine, stromal, lymph, blood, endoderm, ectoderm, and mesoderm. 
     
     
         17 . The tissue construct of  claim 1 , wherein the tissue construct is a vascular wall segment. 
     
     
         18 . An array of living, three-dimensional tissue constructs, each tissue construct comprising: at least one adherent cell type, the at least one adherent cell type cohered and fused to form a living, three-dimensional tissue construct, each tissue construct having a multi-layered architecture, each tissue construct for in vitro use, provided that at least one component of each tissue construct was bioprinted. 
     
     
         19 . The array of  claim 18 , wherein each tissue construct is substantially free of any pre-formed scaffold at the time of bioprinting or the time of use. 
     
     
         20 . The array of  claim 18 , wherein the adherent cells are selected from the group consisting of: liver cells, gastrointestinal cells, pancreatic cells, kidney cells, lung cells, tracheal cells, vascular cells, skeletal muscle cells, cardiac cells, skin cells, smooth muscle cells, connective tissue cells, corneal cells, genitourinary cells, breast cells, reproductive cells, endothelial cells, epithelial cells, fibroblast, neural cells, Schwann cells, adipose cells, bone cells, bone marrow cells, cartilage cells, pericytes, mesothelial cells, cells derived from endocrine tissue, stromal cells, stem cells, progenitor cells, lymph cells, blood cells, endoderm-derived cells, ectoderm-derived cells, mesoderm-derived cells, and combinations thereof. 
     
     
         21 . The array of  claim 18 , wherein each tissue construct within the array is substantially similar. 
     
     
         22 . The array of  claim 18 , wherein one or more of the tissue constructs within the array is unique. 
     
     
         23 . The array of  claim 18 , wherein one or more individual tissues within the array represent human tissues selected from the group consisting of: blood or lymph vessel, muscle, uterus, nerve, mucous membrane, mesothelium, omentum, cornea, skin, liver, kidney, heart, trachea, lung, bone, bone marrow, adipose, connective, bladder, breast, pancreas, spleen, brain, esophagus, stomach, intestine, colon, rectum, ovary, prostate, endocrine tissue, endoderm, mesoderm, and ectoderm. 
     
     
         24 . The array of  claim 18 , wherein each tissue construct exists in a well of a biocompatible multi-well container. 
     
     
         25 . The array of  claim 24 , wherein the wells are coated with one of or more of the following: a biocompatible hydrogel, a protein, a chemical, a peptide, antibodies, or growth factors. 
     
     
         26 . The array of  claim 24 , wherein each tissue construct was placed onto a porous, biocompatible membrane within the wells of the container. 
     
     
         27 . The array of  claim 24 , wherein the container is compatible with an automated or semi-automated drug screening process. 
     
     
         28 . The array of  claim 18 , wherein each tissue construct is secured to a biocompatible surface. 
     
     
         29 . The array of  claim 28 , wherein the biocompatible surface is a porous membrane. 
     
     
         30 . The array of  claim 28 , wherein the biocompatible surface is coated with one of or more of the following: a biocompatible hydrogel, a protein, a chemical, a peptide, antibodies, or growth factors. 
     
     
         31 . The array of  claim 28 , wherein each tissue construct is subjected to shear force, caused by fluid flow, on one or more sides. 
     
     
         32 . The array of  claim 18 , wherein each tissue construct within the array is maintained independently in culture. 
     
     
         33 . The array of  claim 18 , wherein two or more individual tissue constructs within the array exchange soluble factors. 
     
     
         34 . The array of  claim 18 , for use in in vitro assays. 
     
     
         35 . The array of  claim 34 , for use in drug testing. 
     
     
         36 . The array of  claim 18 , wherein at least one tissue within the array is a vascular wall segment. 
     
     
         37 . A living, three-dimensional tissue construct comprising: one or more layers, wherein each layer contains one or more cell types, the one or more layers cohered to form a living, three-dimensional tissue construct, the tissue construct characterized by having at least one of:
 a. 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   b. a plurality of layers, at least one layer compositionally or architecturally distinct from at least one other layer to create a laminar geometry.   
     
     
         38 . The tissue construct of  claim 37 , wherein at least one component of the tissue construct was bioprinted. 
     
     
         39 . The tissue construct of  claim 38 , wherein the tissue construct is substantially free of any pre-formed scaffold at the time of bioprinting or at the time of use. 
     
     
         40 . The tissue construct of  claim 37 , for use in in vitro assays. 
     
     
         41 . The tissue construct of  claim 40 , for use in drug testing. 
     
     
         42 . A method for constructing a living, three-dimensional tissue construct comprising the steps of:
 a. bioprinting bio-ink comprising at least one adherent cell type into or onto a form; and   b. fusing of the bio-ink into a living, three-dimensional tissue construct;   provided that the tissue construct is for in vitro use and not a vascular tube.   
     
     
         43 . The method of  claim 42 , wherein the tissue construct is free of any pre-formed scaffold at the time of bioprinting or the time of use. 
     
     
         44 . The method of  claim 42 , wherein the form is bioprinted. 
     
     
         45 . The method of  claim 44 , wherein the form is bioprinted substantially contemporaneously with the bio-ink. 
     
     
         46 . The method of  claim 42 , further comprising the step of dissolving the form. 
     
     
         47 . A method of constructing a living, three-dimensional tissue construct comprising the steps of:
 a. preparing one or more cohered multicellular aggregates comprising mammalian cells;   b. placing said one or more cohered multicellular aggregates onto a support to form at least one of:
 i. 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 
 ii. a plurality of layers, at least one layer compositionally or architecturally distinct from at least one other layer to create a laminar geometry; 
   c. incubating said one or more multicellular aggregates to allow them to cohere and to form a living, three-dimensional tissue construct.   
     
     
         48 . The method of  claim 47 , wherein at least one component of the tissue construct was bioprinted. 
     
     
         49 . The method of  claim 48 , wherein the tissue construct is free of any pre-formed scaffold at the time of bioprinting or the time of use. 
     
     
         50 . A method of constructing an array of living, three-dimensional tissue constructs comprising the steps of:
 a. preparing cohered multicellular aggregates comprising mammalian cells;   b. placing said cohered multicellular aggregates onto a biocompatible support; wherein said aggregates are spatially arranged in a form suitable for a tissue array; and   c. incubating said multicellular aggregates to allow them to cohere and form an array of living, three-dimensional tissue constructs.   
     
     
         51 . The method of  claim 50 , wherein at least one component of each tissue construct was bioprinted. 
     
     
         52 . The method of  claim 51 , wherein each tissue construct is substantially free of any pre-formed scaffold at the time of bioprinting or the time of use.