Engineered three-dimensional breast tissue, adipose tissue, and tumor disease model

Abstract

Described are three-dimensional, engineered, biological breast tissues, adipose tissues, and tumor models, including breast cancer models.

What is claimed is: 
     
         1 . A three-dimensional, engineered, biological tumor model comprising:
 a. stromal tissue; and   b. tumor tissue;   the tumor tissue comprising cancer cells, the tumor tissue surrounded on all sides by the stromal tissue to form the three-dimensional, engineered, biological tumor model;   provided that the stromal tissue was bioprinted from a stromal bio-ink, the tumor tissue was bioprinted from a tumor bio-ink, or both the stromal tissue and the tumor tissue were bioprinted from their respective bio-inks.   
     
     
         2 . The tumor model of  claim 1 , wherein the model is substantially free of pre-formed scaffold. 
     
     
         3 . The tumor model of  claim 1 , wherein the stromal tissue comprises:
 a. endothelial cells,   b. fibroblasts, and   c. adipocytes, preadipocytes, or both adipocytes and preadipocytes.   
     
     
         4 . The tumor model of  claim 3 , wherein the stromal tissue comprises 55%-75% fibroblasts, 15%-35% endothelial cells, and 1%-20% adipocytes. 
     
     
         5 . The tumor model of  claim 1 , wherein the tumor tissue comprises cells of a cancer cell line. 
     
     
         6 . The tumor model of  claim 1 , wherein the tumor tissue comprises primary cancer cells from a patient tumor. 
     
     
         7 . The tumor model of  claim 1 , wherein the tumor tissue comprises endothelial cells. 
     
     
         8 . The tumor model of  claim 7 , wherein the tumor tissue comprises 65-85% cancer cells and 15%-35% endothelial cells. 
     
     
         9 . The tumor model of  claim 1 , wherein the tumor model is 250 μm to 5 mm in its smallest dimension. 
     
     
         10 . The tumor model of  claim 1 , wherein the stromal tissue is human breast stroma and the tumor tissue is human breast tumor. 
     
     
         11 . The tumor model of  claim 1 , wherein the tumor tissue is completely surrounded on all sides by the stromal tissue to form the three-dimensional, engineered, biological tumor model. 
     
     
         12 . A method of fabricating a three-dimensional, engineered, biological tumor model, the method comprising:
 a. preparing a stromal bio-ink, the stromal bio-ink comprising an extrusion compound and a plurality of stromal cell types;   b. preparing a tumor bio-ink, the tumor bio-ink comprising an extrusion compound and a cancer cell type;   c. depositing the stromal bio-ink and the tumor bio-ink such that the tumor bio-ink is embedded in the stromal bio-ink and in contact with the stromal bio-ink on all sides; and   d. maturing the deposited bio-ink in a cell culture media to remove the extrusion compound and allow the cells to cohere to form a three-dimensional, engineered, biological tumor model.   
     
     
         13 . The method of  claim 12 , wherein the bio-ink is deposited by bioprinting. 
     
     
         14 . The method of  claim 12 , wherein the extrusion compound comprises alginate. 
     
     
         15 . The method of  claim 12 , wherein the extrusion compound is removable by enzymatic digestion. 
     
     
         16 . The method of  claim 15 , wherein the method further comprises crosslinking the deposited bio-ink to physically stabilize the tumor model architecture prior to cohesion of the cells. 
     
     
         17 . The method of  claim 16 , wherein the method further comprises removing the crosslinked bio-ink by enzymatic degradation subsequent to cell cohesion. 
     
     
         18 . The method of  claim 12 , wherein the stromal cell types comprise endothelial cells, fibroblasts and adipocytes or preadipocytes. 
     
     
         19 . The method of  claim 18 , wherein the stromal bio-ink comprises 55%-75% fibroblasts, 15%-35% endothelial cells, and 1%-20% adipocytes. 
     
     
         20 . The method of  claim 12 , wherein the cancer cell type comprises a cancer cell line. 
     
     
         21 . The method of  claim 12 , wherein the cancer cell type comprises primary cancer cells from a patient tumor. 
     
     
         22 . The method of  claim 12 , wherein the tumor bio-ink further comprises endothelial cells. 
     
     
         23 . The method of  claim 12 , wherein the stromal bio-ink comprises 50 million cells per mL to 300 million cells per mL. 
     
     
         24 . The method of  claim 12 , wherein the tumor bio-ink comprises 50 million cells per mL to 300 million cells per mL. 
     
     
         25 . The method of  claim 12 , wherein the cell culture media comprises soluble components that support the grown, maintenance, or differentiation of human fibroblasts, human endothelial cells, adipocytes, and cancer cells. 
     
     
         26 . The method of  claim 12 , wherein depositing the stromal bio-ink and the tumor bio-ink further comprises:
 a. depositing a first sheet of stromal bio-ink on a surface;   b. depositing a continuous border of stromal bio-ink on the first sheet of stromal bio-ink to define a compartment, the compartment open on one side;   c. depositing a node of tumor bio-ink in the compartment; and   d. depositing a second sheet of stromal bio-ink to close the open side of the compartment.   
     
     
         27 . The method of  claim 12 , wherein the tumor model is a breast cancer model. 
     
     
         28 . A three-dimensional, engineered, biological breast cancer model comprising:
 a. breast stromal tissue, the stromal tissue comprising human mammary fibroblasts, human endothelial cells, and human adipocytes; and   b. breast cancer tumor tissue; the tumor tissue comprising breast cancer cells and human endothelial cells, the tumor tissue surrounded on all sides by the stromal tissue to form the three-dimensional, engineered, biological breast cancer model;   provided that the stromal tissue was bioprinted from a stromal bio-ink, the tumor tissue was bioprinted from a tumor bio-ink, or both the stromal tissue and the tumor tissue were bioprinted from their respective bio-inks.   
     
     
         29 . The breast cancer model of  claim 28 , wherein the model is substantially free of pre-formed scaffold. 
     
     
         30 . The breast cancer model of  claim 28 , wherein the stromal tissue comprises 55%-75% fibroblasts, 15%-35% endothelial cells, and 1%-20% adipocytes. 
     
     
         31 . The breast cancer model of  claim 28 , wherein the breast cancer cells are derived from a breast cancer cell line. 
     
     
         32 . The breast cancer model of  claim 28 , wherein the breast cancer cells are primary cancer cells from a patient tumor. 
     
     
         33 . The breast cancer model of  claim 28 , wherein the breast cancer tumor tissue comprises 65-85% cancer cells and 15%-35% endothelial cells. 
     
     
         34 . The breast cancer model of  claim 28 , wherein the breast cancer model is 250 μm to 5 mm in its smallest dimension. 
     
     
         35 . The breast cancer model of  claim 28 , wherein the breast cancer tumor tissue is completely surrounded on all sides by the breast stromal tissue to form the three-dimensional, engineered, biological breast cancer model. 
     
     
         36 . A method of fabricating a three-dimensional, engineered, biological breast cancer model, the method comprising:
 a. preparing a stromal bio-ink, the stromal bio-ink comprising a plurality of stromal cell types, the stromal cell types comprising: an extrusion compound, human mammary fibroblasts, human endothelial cells, and human adipocytes;   b. preparing a tumor bio-ink, the tumor bio-ink comprising: an extrusion compound, a breast cancer cell type and human endothelial cells;   c. depositing the stromal bio-ink and the tumor bio-ink such that the tumor bio-ink is embedded in the stromal bio-ink and in contact with the stromal bio-ink on all sides; and   d. maturing the deposited bio-ink in a cell culture media to remove the extrusion compound allow the cells to cohere to form a three-dimensional, engineered, biological breast cancer model.   
     
     
         37 . The method of  claim 36 , wherein the bio-ink is deposited by bioprinting. 
     
     
         38 . The method of  claim 36 , wherein the extrusion compound comprises alginate. 
     
     
         39 . The method of  claim 36 , wherein the extrusion compound is removable by enzymatic digestion. 
     
     
         40 . The method of  claim 39 , wherein the method further comprises crosslinking the deposited bio-ink to facilitate maintenance of the breast cancer model architecture prior to cohesion of the cells. 
     
     
         41 . The method of  claim 40 , wherein the method further comprises removing the crosslinked bio-ink by enzymatic degradation subsequent to cell cohesion. 
     
     
         42 . The method of  claim 36 , wherein the stromal bio-ink comprises 55%-75% human mammary fibroblasts, 15%-35% human endothelial cells, and 1%-20% human adipocytes. 
     
     
         43 . The method of  claim 36 , wherein the breast cancer cell type comprises a breast cancer cell line. 
     
     
         44 . The method of  claim 36 , wherein the cancer cell type comprises primary breast cancer cells from a patient tumor. 
     
     
         45 . The method of  claim 36 , wherein the stromal bio-ink comprises 50 million cells per mL to 300 million cells per mL. 
     
     
         46 . The method of  claim 36 , wherein the tumor bio-ink comprises 50 million cells per mL to 300 million cells per mL. 
     
     
         47 . The method of  claim 36 , wherein the cell culture media comprises soluble components that support the grown, maintenance, or differentiation of human fibroblasts, human endothelial cells, adipocytes, and cancer cells. 
     
     
         48 . The method of  claim 36 , wherein depositing the stromal bio-ink and the tumor bio-ink further comprises:
 a. depositing a first sheet of stromal bio-ink on a surface;   b. depositing a continuous border of stromal bio-ink on the first sheet of stromal bio-ink to define a compartment, the compartment open on one side;   c. depositing a node of tumor bio-ink in the compartment; and   d. depositing a second sheet of stromal bio-ink to close the open side of the compartment.   
     
     
         49 . The method of  claim 36 , wherein the adipocytes are preadipocytes and the method further comprises providing an adipocyte differentiation signal to the preadipocytes. 
     
     
         50 . A method of identifying a therapeutic agent for cancer in an individual, the method comprising:
 a. preparing a stromal bio-ink, the stromal bio-ink comprising a plurality of stromal cell types;   b. preparing a tumor bio-ink, the tumor bio-ink comprising primary cancer cells from the individual;   c. depositing the stromal bio-ink and the tumor bio-ink such that the tumor bio-ink is embedded in the stromal bio-ink and in contact with the stromal bio-ink on all sides;   d. maturing the deposited bio-ink in a cell culture media to allow the cells to cohere to form a three-dimensional, engineered, biological construct;   e. applying a candidate therapeutic agent to the construct;   f. measuring viability of the cancer cells; and   g. selecting a therapeutic agent for the individual based on the measured viability of the cancer cells;   provided that at least one component of the construct was deposited by bioprinting.   
     
     
         51 . The method of  claim 50 , wherein the stromal bio-ink and the tumor bio-ink are deposited by bioprinting. 
     
     
         52 . The method of  claim 50 , wherein the bio-ink further comprises an extrusion compound. 
     
     
         53 . The method of  claim 50 , wherein the extrusion compound is removable by enzymatic digestion. 
     
     
         54 . The method of  claim 53 , wherein the method further comprises crosslinking the deposited bio-ink to physically stabilize the tumor model architecture prior to cohesion of the cells. 
     
     
         55 . The method of  claim 54 , wherein the method further comprises removing the crosslinked bio-ink by enzymatic degradation subsequent to cell cohesion. 
     
     
         56 . The method of  claim 50 , wherein the stromal cell types comprise endothelial cells, fibroblasts and adipocytes or preadipocytes. 
     
     
         57 . The method of  claim 56 , wherein the stromal bio-ink comprises 55%-75% fibroblasts, 15%-35% endothelial cells, and 1%-20% adipocytes. 
     
     
         58 . The method of  claim 50 , wherein the tumor bio-ink further comprises endothelial cells. 
     
     
         59 . The method of  claim 50 , wherein the stromal bio-ink comprises 50 million cells per mL to 300 million cells per mL. 
     
     
         60 . The method of  claim 50 , wherein the tumor bio-ink comprises 50 million cells per mL to 300 million cells per mL. 
     
     
         61 . The method of  claim 50 , wherein the cell culture media comprises soluble components that support the growth, maintenance, or differentiation of human fibroblasts, human endothelial cells, adipocytes, and cancer cells. 
     
     
         62 . The method of  claim 50 , wherein depositing the stromal bio-ink and the tumor bio-ink further comprises:
 a. depositing a first sheet of stromal bio-ink on a surface;   b. depositing a continuous border of stromal bio-ink on the first sheet of stromal bio-ink to define a compartment, the compartment open on one side;   c. depositing a node of tumor bio-ink in the compartment; and   d. depositing a second sheet of stromal bio-ink to close the open side of the compartment.   
     
     
         63 . The method of  claim 50 , wherein the three-dimensional, engineered, biological construct is a breast cancer construct. 
     
     
         64 . A three-dimensional, engineered, biological tissue comprising viable, differentiated adipocytes. 
     
     
         65 . The tissue of  claim 64 , wherein the tissue is bioprinted. 
     
     
         66 . The tissue of  claim 65 , wherein the tissue is substantially free of pre-formed scaffold. 
     
     
         67 . The tissue of  claim 64 , wherein the tissue comprises at least 5% viable, differentiated adipocytes. 
     
     
         68 . The tissue of  claim 67 , wherein the tissue comprises at least 10% viable, differentiated adipocytes. 
     
     
         69 . The tissue of  claim 64 , wherein at least 50% of the adipocytes are viable 24 hours post-fabrication. 
     
     
         70 . The tissue of  claim 69 , wherein at least 75% of the adipocytes are viable 24 hours post-fabrication. 
     
     
         71 . The tissue of  claim 64 , wherein the adipocytes secrete leptin for at least 1 week post-fabrication. 
     
     
         72 . The tissue of  claim 64 , wherein the tissue is adipose tissue. 
     
     
         73 . The tissue of  claim 64 , wherein the adipocytes are subcutaneous adipocytes. 
     
     
         74 . The tissue of  claim 64 , wherein the adipocytes are derived from preadipocytes or mesenchymal stem cells. 
     
     
         75 . A method of fabricating a three-dimensional, engineered, adipose tissue-containing, biological construct, the method comprising:
 a. providing an adipocyte differentiation signal to preadipocytes;   b. preparing a preadipocyte bio-ink, the bio-ink comprising the preadipocytes and at least one other cell type;   c. depositing the bio-ink on a surface; and   d. maturing the bio-ink in a cell culture media to allow the cells to cohere to form a three-dimensional, engineered, biological construct, the construct comprising viable, differentiated adipocytes.   
     
     
         76 . The method of  claim 75 , wherein the bio-ink is deposited by bioprinting. 
     
     
         77 . The method of  claim 75 , wherein the construct comprises at least 5% viable, differentiated adipocytes. 
     
     
         78 . The method of  claim 77 , wherein the construct comprises at least 10% viable, differentiated adipocytes. 
     
     
         79 . The method of  claim 75 , wherein at least 50% of the adipocytes are viable 24 hours post-fabrication. 
     
     
         80 . The method of  claim 79 , wherein at least 75% of the adipocytes are viable 24 hours post-fabrication. 
     
     
         81 . The method of  claim 75 , wherein the adipocytes secrete leptin for at least 1 week post-fabrication. 
     
     
         82 . The method of  claim 75 , wherein the construct is adipose tissue. 
     
     
         83 . The method of  claim 75 , wherein the preadipocytes are subcutaneous preadipocytes. 
     
     
         84 . A three-dimensional, engineered, biological breast tissue comprising: human mammary fibroblasts, human endothelial cells, human mammary epithelial cells, and human adipocytes; provided that the cells were bioprinted from a bio-ink and cohered to form the three-dimensional, engineered, biological breast tissue; provided that the tissue is substantially free of pre-formed scaffold. 
     
     
         85 . The breast tissue of  claim 84 , wherein the tissue comprises 55%-75% fibroblasts, 15%-35% endothelial cells, and 1%-20% adipocytes. 
     
     
         86 . The breast tissue of  claim 84 , wherein the tissue is 250 μm to 5 mm in its smallest dimension. 
     
     
         87 . The breast tissue of  claim 84 , wherein the tissue is exposed to a disease-causing agent to create a breast tissue disease model. 
     
     
         88 . The breast tissue of  claim 87 , wherein the disease-causing agent comprises a virus, a bacterium, a chemical compound, or a combination thereof. 
     
     
         89 . A method of fabricating a three-dimensional, engineered, biological breast tissue, the method comprising:
 a. providing an adipocyte differentiation signal to human preadipocytes;   b. preparing a bio-ink, the bio-ink comprising a plurality of breast cell types, the breast cell types comprising human mammary fibroblasts, human endothelial cells, human mammary epithelial cells, and the human preadipocytes;   c. depositing the bio-ink on a biocompatible surface; and   d. maturing the deposited bio-ink in a cell culture media to allow the cells to cohere to form a three-dimensional, engineered, biological breast tissue.   
     
     
         90 . The method of  claim 89 , wherein the bio-ink is deposited by bioprinting. 
     
     
         91 . The method of  claim 89 , wherein the bio-ink comprises 55%-75% human mammary fibroblasts, 15%-35% human endothelial cells, and 1%-20% human preadipocytes. 
     
     
         92 . The method of  claim 89 , wherein the bio-ink comprises 50 million cells per mL to 300 million cells per mL. 
     
     
         93 . The method of  claim 89 , wherein the cell culture media comprises soluble components that support the growth, maintenance, or differentiation of human fibroblasts, human endothelial cells, and adipocytes. 
     
     
         94 . The method of  claim 89 , wherein the method further comprises exposing the three-dimensional, engineered, biological breast tissue to a disease-causing agent to create a breast tissue disease model. 
     
     
         95 . The method of  claim 94 , wherein the disease-causing agent comprises a virus, a bacterium, a chemical compound, or a combination thereof. 
     
     
         96 . An array of three-dimensional, engineered, biological tumor models, each tumor model comprising: stromal tissue and tumor tissue; the tumor tissue comprising cancer cells, the tumor tissue surrounded on all sides by the stromal tissue to form each three-dimensional, engineered, biological tumor model; provided that the stromal tissue, the tumor tissue, or both the stromal tissue and the tumor tissue were bioprinted; provided that the array is adapted for use in a high throughput assay. 
     
     
         97 . The array of  claim 96 , wherein each tumor model is substantially free of pre-formed scaffold. 
     
     
         98 . The array of  claim 96 , wherein each tumor model is in a well of a multi-well plate. 
     
     
         99 . The array of  claim 96 , wherein the stromal tissue comprises: endothelial cells, fibroblasts, and adipocytes, preadipocytes, or both adipocytes and preadipocytes. 
     
     
         100 . The array of  claim 99 , wherein the stromal tissue comprises 55%-75% fibroblasts, 15%-35% endothelial cells, and 1%-20% adipocytes. 
     
     
         101 . The array of  claim 96 , wherein the tumor tissue comprises primary cancer cells from a patient tumor. 
     
     
         102 . The array of  claim 96 , wherein the tumor tissue comprises endothelial cells. 
     
     
         103 . The array of  claim 102 , wherein the tumor tissue comprises 65-85% cancer cells and 15%-35% endothelial cells. 
     
     
         104 . The array of  claim 96 , wherein each tumor model is 250 μm to 5 mm in its smallest dimension. 
     
     
         105 . The array of  claim 96 , wherein the stromal tissue is human breast stroma and the tumor tissue is human breast tumor. 
     
     
         106 . The array of  claim 96 , wherein the tumor tissue is completely surrounded on all sides by the stromal tissue to form each three-dimensional, engineered, biological tumor model. 
     
     
         107 . An array of three-dimensional, engineered, biological breast cancer models, each breast cancer model comprising:
 a. stromal tissue, the stromal tissue comprising human mammary fibroblasts, human endothelial cells, and human adipocytes; and   b. tumor tissue; the tumor tissue comprising breast cancer cells and human endothelial cells, the tumor tissue surrounded on all sides by the stromal tissue to form each three-dimensional, engineered, biological breast cancer model;   provided that the stromal tissue, the tumor tissue, or both the stromal tissue and the tumor tissue were bioprinted; provided that the array is adapted for use in a high throughput assay.   
     
     
         108 . The array of  claim 107 , wherein each breast cancer model is substantially free of pre-formed scaffold. 
     
     
         109 . The array of  claim 107 , wherein each breast cancer model is in a well of a multi-well plate. 
     
     
         110 . The array of  claim 107 , wherein the stromal tissue comprises 55%-75% fibroblasts, 15%-35% endothelial cells, and 1%-20% adipocytes. 
     
     
         111 . The array of  claim 107 , wherein the breast cancer cells are primary cancer cells from a patient tumor. 
     
     
         112 . The array of  claim 107 , wherein the tumor tissue comprises 65-85% cancer cells and 15%-35% endothelial cells. 
     
     
         113 . The array of  claim 107 , wherein each breast cancer model is 250 μm to 5 mm in its smallest dimension. 
     
     
         114 . The array of  claim 107 , wherein the tumor tissue is completely surrounded on all sides by the stromal tissue to form each three-dimensional, engineered, biological breast cancer model. 
     
     
         115 . A three-dimensional, engineered, biological tumor tissue comprising human cancer cells; provided that the cells are cohered to form the three-dimensional, engineered, biological tumor tissue; provided that the tumor tissue is substantially free of pre-formed scaffold. 
     
     
         116 . The tumor tissue of  claim 115 , wherein the tissue was bioprinted from a cancer call bio-ink. 
     
     
         117 . The tumor tissue of  claim 115 , wherein the tissue further comprises one or more of fibroblasts, endothelial cells, epithelial cells, adipocytes, and immune cells. 
     
     
         118 . The tumor tissue of  claim 115 , wherein the tissue is 250 μm to 5 mm in its smallest dimension. 
     
     
         119 . The tumor tissue of  claim 115 , wherein the tissue was exposed to a carcinogen to transform the cells. 
     
     
         120 . The breast tissue of  claim 119 , wherein the disease-causing agent comprises a virus, a bacterium, a chemical compound, or a combination thereof. 
     
     
         121 . The tumor tissue of  claim 115 , wherein a crosslinkable extrusion compound is used to physically stabilize the tumor tissue subsequent to fabrication and prior to cohesion of the cells to form the tumor tissue.