HK1216651B - Engineered liver tissues, arrays thereof, and methods of making the same - Google Patents

Description

Engineered liver tissue, its array and preparation method thereof

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and is a continuation-in-part of U.S. application serial number 13/841,430, filed March 15, 2013, which is incorporated herein by reference in its entirety.

Background Art

The healthcare industry faces many pressing issues. As of June 2012, 114,636 patients were registered with the United Network for Organ Sharing (UNOS) for organ transplantation. According to UNOS, only 6,838 transplants were performed between January and March 2012. Each year, more patients are added to the UNOS list than transplants are performed, resulting in a net increase in the number of patients waiting for a transplant.

In addition, the research and development cost of a new drug compound is approximately $1.8 billion. See Paul et al. (2010). How to improve R&D productivity: the pharmaceutical industry's grand challenge, Nature Reviews Drug Discovery 9(3): 203-214. Drug discovery is the process of discovering and/or designing drugs. The drug discovery process generally includes at least the following steps: candidate identification, synthesis, characterization, screening, and analysis of therapeutic effects. Despite advances in technology and understanding of biological systems, drug discovery remains a lengthy, expensive, and inefficient process, and the discovery rate of new therapeutic agents is low.

Summary of the Invention

The present invention relates to the fields of regenerative medicine and tissue and/or organ engineering. More specifically, the present invention relates to engineered liver tissue constructs, arrays thereof, and methods of making the same.

In one aspect, disclosed herein is an engineered, living three-dimensional liver tissue construct comprising at least one compartment defining a planar geometry, the compartment comprising an interior defined by a border, the interior comprising parenchymal cells, the border comprising non-parenchymal cells; the cells condense to form a living three-dimensional liver tissue construct; provided that at least a portion of the construct is bioprinted and the construct is substantially free of preformed scaffolds when in use. In some embodiments, the construct further comprises an extrusion compound that improves the suitability of the cells for bioprinting. In some embodiments, the parenchymal cells are derived from one or more of the following sources: adult mammalian liver tissue; fetal mammalian liver tissue; established liver-derived cell lines or strains, embryonic stem cells (ESCs); ESC-derived hepatocyte-like cells, induced pluripotent stem cells (iPSCs); iPSC-derived hepatocyte-like cells; adult stem cells/progenitor cells derived from the liver; and adult stem cells/progenitor cells derived from non-liver tissues. In some embodiments, the non-parenchymal cells include one or more of the following: vascular cells, endothelial cells, fibroblasts, mesenchymal cells, immune cells, Kupffer cells, astrocytes, bile duct epithelial cells, bile duct epithelial-like cells, sinusoidal endothelial cells, stem cells/progenitor cells derived from the liver, and stem cells/progenitor cells not derived from the liver. In some embodiments, the construct comprises stem cells/progenitor cells exposed to one or more differentiation signals. In further embodiments, the differentiation signals include one or more of the following: biomechanical signals, soluble signals, and physical signals. In further embodiments, the stem cells/progenitor cells are exposed to one or more differentiation signals before construct manufacture. In further embodiments, the stem cells/progenitor cells are exposed to one or more differentiation signals during construct manufacture. In further embodiments, the stem cells/progenitor cells are exposed to one or more differentiation signals after construct manufacture. In some embodiments, the construct comprises one or more layers. In further embodiments, the construct comprises multiple layers, at least one layer being compositionally or structurally different from at least one other layer to create a layered geometry. In some embodiments, the construct is used for in vitro analysis. In some embodiments, the construct is used for one or more of the following: drug discovery; drug testing; preclinical studies; toxicity testing; absorption, distribution, metabolism and excretion tests (ADME); drug metabolism and pharmacokinetic tests (DMPK); disease modeling; infectious disease modeling; host disease modeling; three-dimensional biological research; and cell-based screening. In some embodiments, the construct is used for cell-based screening, wherein the screening is used to screen one or more infectious diseases, liver fibrosis (e.g., cirrhosis), liver cancer, liver steatosis (e.g., fatty liver), one or more metabolic defects or one or more protein deficiencies. In further embodiments, the infectious disease includes viral infection or parasitic infection (e.g., Plasmodium infection, etc.). In some embodiments, the construct is used for long-term tissue toxicity studies, wherein the analysis is performed with a cycle of>3 days and up to 6 months. In some embodiments, the construct is used to strengthen one or more liver functions in the human body. In further embodiments, the construct is used to implant the damage, lesion or degeneration (degeneration) site in the subject. In further embodiments, the construct is for clinical use in an in vitro device designed to enhance or restore one or more liver functions. In some embodiments, the construct is non-innervated.

In another aspect, disclosed herein is an engineered, living three-dimensional liver tissue construct comprising: one or more layers, each layer comprising one or more liver cell types, the one or more layers condensing to form a living three-dimensional liver tissue construct, the construct being characterized by having at least one of the following: at least one layer comprising multiple cell types, the cell types spatially arranged relative to each other to create a planar geometry; and multiple layers, at least one layer being compositionally or structurally different from at least one other layer to create a layered geometry. In some embodiments, at least a portion of the construct is bioprinted. In some embodiments, each layer of the engineered liver tissue comprises multiple cells in the X, Y, and Z axes. In some embodiments, each layer of the engineered liver tissue has a thickness in the X, Y, and Z axes of at least about 50 microns. In further embodiments, the construct further comprises an extrusion compound that improves the suitability of the cells for bioprinting. In some embodiments, the construct is substantially free of preformed scaffolds when in use. In some embodiments, the hepatocytes 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 cells/progenitor cells derived from the liver; and adult stem cells/progenitor cells derived from non-liver tissues. In some embodiments, the construct further comprises 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, astrocytes, bile duct cells, sinusoidal endothelial cells, stem cells/progenitor cells derived from the liver, and stem cells/progenitor cells not derived from the liver. In some embodiments, the construct comprises stem cells/progenitor cells exposed to one or more differentiation signals. In further embodiments, the differentiation signals include one or more of the following: biomechanical signals, resolvable/soluble signals, and physical signals. In further embodiments, the stem cells/progenitor cells are exposed to one or more differentiation signals before construct manufacture. In a further embodiment, the stem cell/progenitor cell is exposed to one or more differentiation signals during the manufacture of the construct. In a further embodiment, the stem cell/progenitor cell is exposed to one or more differentiation signals after the manufacture of the construct. In some embodiments, the construct is used for in vitro analysis. In a further embodiment, the construct is used for one or more of the following: drug discovery; drug testing; preclinical studies; toxicity testing; absorption, distribution, metabolism and excretion tests (ADME); drug metabolism and pharmacokinetic tests (DMPK); disease modeling; infectious disease modeling; host disease modeling; three-dimensional biological research; and cell-based screening. In some embodiments, the construct is used for cell-based screening, wherein the screening is used to screen one or more infectious diseases, liver fibrosis (e.g., cirrhosis), liver cancer, liver steatosis (e.g., fatty liver), one or more metabolic defects or one or more protein deficiencies. In a further embodiment, the infectious disease includes viral infection or parasitic infection (e.g., Plasmodium infection, etc.). In some embodiments, the construct is used for long-term tissue toxicity studies, wherein the analysis is carried out in a cycle of> 3 days and up to 6 months. In some embodiments, the construct is used to strengthen one or more liver functions in the human body. In further embodiments, the construct is for implantation into a subject at a site of injury, disease, or degeneration. In further embodiments, the construct is for clinical use in an in vitro device designed to enhance or restore one or more liver functions. In some embodiments, the construct is non-innervated.

On the other hand, disclosed herein is an engineered, living three-dimensional liver tissue construct comprising: a plurality of layers, each layer comprising a cylindrical bio-ink, the bio-ink being aligned substantially parallel to the axis, the bio-ink comprising parenchymal hepatocytes; and optionally, non-parenchymal hepatocytes within or between the cylindrical bio-inks; and optionally, void spaces between the cylindrical bio-inks. In some embodiments, at least a portion of the construct is bioprinted. In further embodiments, the construct further comprises an extrusion compound that improves the suitability of the cells for bioprinting. In some embodiments, 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); ESC-derived hepatocyte-like cells, induced pluripotent stem cells (iPSC); iPSC-derived hepatocyte-like cells; adult stem cells/progenitor cells derived from the liver; and adult stem cells/progenitor cells derived from non-liver tissue. In some embodiments, the non-parenchymal cells include one or more of the following: vascular cells, endothelial cells, fibroblasts, mesenchymal cells, immune cells, Kupffer cells, astrocytes, bile duct epithelial cells, bile duct epithelial-like cells, sinusoidal endothelial cells, stem cells/progenitor cells derived from the liver, and stem cells/progenitor cells not derived from the liver. In some embodiments, the construct comprises one or more layers. In further embodiments, the construct comprises multiple layers, at least one layer being different in composition or structure from at least one other layer to create a layered geometry. In some embodiments, the construct is used to enhance one or more liver functions in the human body. In further embodiments, the construct is used to implant an injury, lesion, or degenerative site in a subject. In further embodiments, the construct is used for clinical use in an in vitro device designed to enhance or restore one or more liver functions. In some embodiments, the construct is not innervated by nerves.

In another aspect, disclosed herein are arrays of engineered, living, three-dimensional liver tissue constructs, each construct comprising: one or more layers, each layer comprising one or more liver cell types, the one or more layers condensing to form a living, three-dimensional liver tissue construct, each construct being characterized by having at least one of the following: at least one layer comprising a plurality of cell types spatially arranged relative to one another to create a planar geometry; and a plurality of layers, at least one layer being compositionally or structurally different from at least one other layer to create a layered geometry. In some embodiments, at least a portion of each construct is bioprinted. In further embodiments, each construct further comprises an extrusion compound that improves the suitability of the cells for bioprinting. In some embodiments, each construct is substantially free of a preformed scaffold when used. In some embodiments, 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 cells/progenitor cells derived from the liver; and adult stem cells/progenitor cells derived from non-liver tissue. In some embodiments, each construct comprises stem cells/progenitor cells exposed to one or more differentiation signals. In further embodiments, the differentiation signals include one or more of the following: biomechanical signals, resolvable/soluble signals, and physical signals. In further embodiments, the stem cells/progenitor cells are exposed to one or more differentiation signals before construct manufacture. In further embodiments, the stem cells/progenitor cells are exposed to one or more differentiation signals during construct manufacture. In further embodiments, the stem cells/progenitor cells are exposed to one or more differentiation signals after construct manufacture. In some embodiments, each construct further comprises 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, astrocytes, bile duct cells, sinusoidal endothelial cells, stem cells/progenitor cells derived from the liver, and stem cells/progenitor cells not derived from the liver. In some embodiments, the array is used for in vitro analysis. In further embodiments, the array is used for one or more of the following: drug discovery; drug testing; preclinical studies; toxicity testing; absorption, distribution, metabolism and excretion assays (ADME); drug metabolism and pharmacokinetic assays (DMPK); disease modeling; infectious disease modeling; host disease modeling; three-dimensional biological studies; and cell-based screening. In some embodiments, the array is used for cell-based screening, wherein the screening is for screening one or more infectious diseases, liver fibrosis (e.g., cirrhosis), liver cancer, liver steatosis (e.g., fatty liver), one or more metabolic defects or one or more protein deficiencies. In further embodiments, the infectious disease includes viral infection or parasitic infection (e.g., Plasmodium infection, etc.). In some embodiments, the array is used for long-term tissue toxicity studies, wherein the analysis is performed over a period of>3 days and up to 6 months.

In another aspect, disclosed herein are arrays of engineered, living, three-dimensional tissue constructs, wherein at least one of the constructs is a liver tissue construct, each liver tissue construct comprising: one or more layers, each layer comprising one or more liver cell types, the one or more layers cohesing to form a living, three-dimensional liver tissue construct, the liver tissue construct being characterized by having at least one of the following: at least one layer comprising a plurality of cell types spatially arranged relative to one another to create a planar geometry; and a plurality of layers, at least one layer being compositionally or structurally different from at least one other layer to create a layered geometry. In some embodiments, at least a portion of each liver tissue construct is bioprinted. In further embodiments, each liver tissue construct further comprises an extrusion compound that improves the suitability of the cells for bioprinting. In some embodiments, each liver construct is substantially free of a preformed scaffold when in use. In some embodiments, the hepatocytes 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 cells/progenitor cells derived from the liver; and adult stem cells/progenitor cells derived from non-liver tissues. In some embodiments, each liver tissue construct comprises stem cells/progenitor cells exposed to one or more differentiation signals. In further embodiments, the differentiation signals include one or more of the following: biomechanical signals, resolvable/soluble signals, and physical signals. In further embodiments, the stem cells/progenitor cells are exposed to one or more differentiation signals before construct manufacture. In further embodiments, the stem cells/progenitor cells are exposed to one or more differentiation signals during construct manufacture. In further embodiments, the stem cells/progenitor cells are exposed to one or more differentiation signals after construct manufacture. In some embodiments, each liver tissue construct further comprises 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, astrocytes, bile duct cells, sinusoidal endothelial cells, stem cells/progenitor cells derived from the liver, and stem cells/progenitor cells not derived from the liver. In some embodiments, the array is used for in vitro analysis. In further embodiments, the array is used for one or more of the following: tissue-tissue interaction; drug discovery; drug testing; preclinical studies; toxicity testing; absorption, distribution, metabolism, and excretion testing (ADME); drug metabolism and pharmacokinetic testing (DMPK); disease modeling; infectious disease modeling; host disease modeling; three-dimensional biological studies; and cell-based screening. In some embodiments, the array is used for cell-based screening, wherein the screening is used to screen for one or more infectious diseases, liver fibrosis (e.g., cirrhosis), liver cancer, liver steatosis (e.g., fatty liver), one or more metabolic defects, or one or more protein deficiencies. In further embodiments, the infectious disease includes viral infection or parasitic infection (e.g., Plasmodium infection, etc.). In some embodiments, the array is used for long-term tissue toxicity studies, where analysis is performed over a period of >3 days and up to 6 months.

In another aspect, disclosed herein is a method for making a living three-dimensional liver tissue construct, the method comprising: preparing one or more bio-inks comprising non-parenchymal cells; preparing one or more bio-inks comprising parenchymal cells; depositing the bio-inks onto a support; and incubating the deposited bio-inks for about 1 hour to about 30 days to form a living three-dimensional liver tissue construct comprising at least one compartment comprising an interior comprising parenchymal cells bounded by a periphery comprising non-parenchymal cells. In some embodiments, the non-parenchymal cells include one or more of the following: vascular cells, endothelial cells, fibroblasts, mesenchymal cells, immune cells, cancer cells, Kupffer cells, astrocytes, bile duct epithelial cells, bile duct epithelial-like cells, sinusoidal endothelial cells, liver-derived stem/progenitor cells, and non-liver-derived stem/progenitor cells. In some embodiments, 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); ESC-derived hepatocyte-like cells, induced pluripotent stem cells (iPSC); iPSC-derived hepatocyte-like cells; adult stem cells/progenitor cells derived from the liver; and adult stem cells/progenitor cells derived from non-liver tissue. In some embodiments, at least one portion of the construct is bioprinted. In further embodiments, the construct further comprises an extrusion compound that improves the suitability of the cells for bioprinting. In some embodiments, the construct is substantially free of any preformed scaffold when in use. In some embodiments, the construct is not innervated by nerves.

In another aspect, disclosed herein is a method for making an engineered, living three-dimensional liver tissue construct, the method comprising: preparing one or more bio-inks comprising hepatocytes; depositing the one or more bio-inks onto a support; and incubating the one or more deposited bio-inks for 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 condensing to form a living three-dimensional liver tissue construct. In some embodiments, the hepatocytes are derived from one or more of the following sources: adult mammalian liver tissue; fetal mammalian liver tissue; embryonic stem cells (ESCs); ESC-derived hepatocyte-like cells, induced pluripotent stem cells (iPSCs); iPSC-derived hepatocyte-like cells; adult stem cells/progenitor cells derived from the liver; and adult stem cells/progenitor cells derived from non-liver tissue. In some embodiments, the construct further comprises 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, astrocytes, bile duct cells, sinusoidal endothelial cells, stem/progenitor cells derived from the liver, and stem/progenitor cells not derived from the liver. In some embodiments, the construct is characterized by having at least one of the following: at least one layer comprising multiple cell types, the cell types spatially arranged relative to each other to create a planar geometry; and multiple layers, at least one layer being compositionally or structurally different from at least one other layer to create a layered geometry. In some embodiments, at least a portion of the construct is bioprinted. In further embodiments, the construct further comprises an extrusion compound that improves the suitability of the cells for bioprinting. In some embodiments, the construct is substantially free of any preformed scaffold when in use. In some embodiments, the construct is not innervated.

In another aspect, disclosed herein is a method for constructing a living three-dimensional liver tissue construct, the method comprising: preparing one or more cohesive multicellular aggregates comprising mammalian liver cells; placing the one or more cohesive multicellular aggregates on a support to form at least one of the following: at least one layer comprising a plurality of cell types spatially arranged relative to each other to create a planar geometry; and a plurality of layers, at least one layer being compositionally or structurally different from at least one other layer to create a layered geometry; and incubating the one or more multicellular aggregates for about 1 hour to about 30 days to allow them to cohere and form a living three-dimensional liver tissue construct. In some embodiments, 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 cells/progenitor cells derived from the liver; and adult stem cells/progenitor cells derived from non-liver tissue. In some embodiments, the liver tissue construct further comprises 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, astrocytes, bile duct cells, sinusoidal endothelial cells, stem/progenitor cells derived from the liver, and stem/progenitor cells not derived from the liver. In some embodiments, at least a portion of the construct is bioprinted. In some embodiments, the construct further comprises an extrusion compound that improves the suitability of the cells for bioprinting. In some embodiments, the construct does not contain any preformed scaffold at the time of bioprinting or use. In some embodiments, the construct is not innervated.

In another aspect, disclosed herein is a method for making a living three-dimensional liver tissue construct, the method comprising incubating one or more bio-inks comprising parenchymal cells and one or more bio-inks comprising non-parenchymal cells deposited onto a support for a period of about 1 hour to about 30 days to form a living three-dimensional liver tissue construct, the construct comprising at least one compartment comprising an interior comprising parenchymal cells bounded by a side comprising non-parenchymal cells.

In another aspect, disclosed herein is an engineered, living, three-dimensional liver tissue construct comprising one or more layers, at least one layer comprising at least two cell types, the at least two cell types comprising a parenchymal liver cell type and a non-parenchymal liver cell type, the at least two cell types spatially arranged relative to each other to create a planar geometry comprising at least one parenchymal liver cell compartment and at least one non-parenchymal liver cell compartment, the one or more layers cohesing to form a living, three-dimensional liver tissue construct, provided that the living, three-dimensional liver tissue construct remains substantially viable in in vitro culture for at least 7 days and possesses at least two of the following liver characteristics: synthesis of cholesterol; storage of lipids; storage of glycogen; expression of liver-specific proteins comprising two or more of the following: albumin, fibrinogen, transferrin, CYP450, α-1-antitrypsin, ornithine transcarbamylase, glycogen synthase, factor VIII and factor IX; responsive to liver regulators, including one or more of the following: hormones, inducers, toxins, drugs, antibodies, interfering RNA, small RNA, locked nucleic acid, viruses, bacteria, parasites and inflammatory inducers; responsive to liver toxicants, including one or more of the following: acetaminophen, alcohol, trovafloxacin, methotrexate, diclofenac, troglitazone, valproic acid, amiodarone; express transporters, including one or more of the following: BSEP, OATP1B1, NTCP, PGP, BCRP, OATP1B3, ABCB4, ATP8B1, multidrug resistance transporter (MDR); maintain at least some quiescent astrocytes after manufacturing; and contain microvascular structures. In some embodiments, the construct remains substantially viable for at least 14 days in in vitro culture. In some embodiments, the construct remains substantially viable for at least 28 days in in vitro culture. In a further embodiment, the construct remains substantially alive for at least 40 days in vitro culture. In various embodiments, the construct remains substantially alive for at least 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more days in vitro culture. In some embodiments, the construct is produced using a bioprinter with a fully automatic process. In some embodiments, the at least two cell types include cells derived from induced pluripotent stem cells (iPSC). In some embodiments, the at least two cell types include myeloid cells derived from monocytes or macrophages. In various embodiments, the construct has at least three, four, five or six, seven, eight or all of the liver features.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the present invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description of the invention which sets forth illustrative embodiments in which the principles of the invention are utilized and the accompanying drawings, in which:

FIG1 is a non-limiting, exemplary illustration of native liver tissue geometry.

FIG2 is a non-limiting, exemplary demonstration of the maturation and biochemical characterization of bioprinted liver constructs containing HepaRG cells. HepaRG cells were deposited in the center of a perimeter box and then matured for 96 hours (A-C). The bioprinted liver constructs were metabolically active, producing albumin (D) and cholesterol (E) at 96 hours. After dexamethasone treatment, cytochrome P450 (CYP3A4) activity was 5-fold higher than that of untreated controls (F).

FIG3 is a non-limiting example of structures bioprinted in defined geometric patterns (A, B) containing iPSC-derived hepatocytes (C) that synthesize significantly higher levels of albumin (D) (per million input cells) in the engineered, bioprinted constructs than in 2D culture controls.

Figure 4 is a non-limiting, exemplary demonstration of the maturation of a bioprinted liver construct (A) containing discrete spheroids of non-parenchymal cells (B). The bioprinted construct remained confluent and stable after an additional 24 hours of incubation (C, D).

FIG5 is a non-limiting, exemplary demonstration of the dissolution of a co-printed mold, which allows for spatial and temporal control over the printing of hepatocytes and results in the generation of user-specified compartments of defined shape and size in the x, y, and z axes. Arrows indicate areas where the co-printed mold has dissolved 24 hours after printing and where additional cell input can be added.

Figure 6 depicts macroscopic images of non-limiting examples of engineered liver tissue, in this case, multilayered liver tissue bioprinted using a continuous deposition mechanism using a bioink composed of multiple liver cell types and a water-soluble extrusion compound (e.g., PF-127). (A) shows a schematic of a single functional unit, highlighting the planar geometry constructed by patterning the bioink and negative space; (B) a tessellated functional unit bioprinted with PF-127 containing 2 x ¹⁰⁸ cells; (C) shows the construct after 20 minutes of applying culture medium; and (D) shows the construct after applying culture medium to the structure and dissolving the extrusion compound for 16 hours. The degree of retention of the planar geometry over time was recorded.

Figure 7 is a non-limiting photomicrograph of the mosaic construct of Figure 6, depicting exemplary "spokes" in the mosaic construct. The photomicrograph shows hematoxylin and eosin staining of formalin-fixed paraffin-embedded tissue sections of astrocytes, endothelial cells, and dermal fibroblasts bioprinted by serial deposition in multi-layered mosaic hexagonal structures and then cultured for 16 hours.

Figure 8 is a non-limiting example of the dissolution of a co-printed mold, which allows the fusion of different compartmentalized hepatocyte regions (marked by arrows). The regions fuse into solid bioprinted tissue over time (B; T = 24 hours).

Figure 9 is a non-limiting example of a co-printed mold (4% gelatin and 2% alginate) bioprinted with the syringe deposition module (SDM) of the MMX Bioprinter to produce a construct (A) that fused and matured into liver tissue (B) 72 hours after printing. In this example, the bioprinted tissue was dense as demonstrated by H&E staining (C) and viable as demonstrated by the relatively few TUNEL-stained cells (D) compared to proliferating cells (E).

Figure 10 is a non-limiting example of a co-printed mold (A; 4% gelatin and 2% alginate) that was bioprinted by continuous deposition with a syringe deposition module (SDM) to produce a confluent three-dimensional liver tissue construct. Adding endothelial bioink to the center of the structure increased the complexity of the construct (B). E-cadherin (C) and CD31 (D) staining showed that 144 hours after printing, epithelial and endothelial cells were prevalent in the area where they were bioprinted. TUNEL staining showed limited cell death within the core of the engineered liver tissue after 144 hours of incubation (E).

Figure 11 is a non-limiting example of a co-printed mold (co-mold) composed of a hydrogel (4% gelatin: 2% alginate) (A) or a bioink composed of non-parenchymal cells and a hydrogel-based extruded compound (B). The bioink co-mold line contained 150 x ¹⁰⁶ cells per mL of hydrogel extruded compound. HepG2 cells were added to the co-mold structure and incubated for 24 hours, resulting in a confluent, bioprinted liver structure (C). Dissolution of the extruded compound occurred over time in the aqueous medium.

Figure 12 is a non-limiting example of dense cellular liver tissue produced by sequential deposition using two syringe deposition modules (SDM) on a NovoGen MMX Bioprinter. A common mold line containing 4% gelatin and 2% alginate was deposited by the first SDM module and subsequently filled with HepG2 bioslurry using the second SDM.

Figure 13 is a non-limiting example of a bioprinted neotissue with a layered geometry. A NovoGel ^™ hydrogel substrate and a co-printed confining box were bioprinted, followed by the deposition of a first layer containing hepatic epithelial cell bioink (HepG2 cells), onto which a second layer consisting of hepatic stellate cells and endothelial cells was bioprinted. In this example, the stellate cell:EC layer was bioprinted by continuously depositing a bioink containing a hydrogel extrusion compound (A). An overall image of the construct obtained immediately after fabrication shows two distinct bioink layers (B). Hematoxylin and eosin staining (C) of sections of formalin-fixed, paraffin-embedded constructs after 48 hours of culture shows the different morphologies of the two layers and the establishment of a layered geometry. CD31-positive cells were confined to the upper layer of the construct, in which a suspension of endothelial cells and hepatic stellate cells was bioprinted (D), while IGF-2-positive HepG2 was only found in the bottom layer (E).

Figure 14 is a non-limiting example of cell patterning and layering in bioprinted liver tissue. Hematoxylin and eosin staining of paraffin-embedded tissue sections shows adjacent new tissue (A) formed by bioprinting a polymorphic cell population containing vascular endothelial cells and hepatic stellate cells. Staining of tissue sections with CD31 antibodies shows the presence of centrally located microvessels lined with ECs and an outer layer of CD31-positive ECs (B).

Figure 15 is a non-limiting example of stimulation of bioprinted hepatic stellate cell tissue with TGF-β1. Incubation of bioprinted hepatic stellate cell sheets with increasing concentrations of TGF-β1 (0, 1, 10, 50 ng/mL) resulted in gross changes in the bioprinted tissue, with increasing cytokine concentrations leading to increased tissue outgrowth formation (A-D, 0-50 ng/mL). Trichrome staining of tissue sections from bioprinted hepatic stellate cell-containing tissue showed increases in collagen deposition and construct size, as well as a significant decrease in cell density (E-H, 0-50 ng/mL).

Figure 16 shows a non-limiting example of bioprinting in a multi-well format. Bioprinted tissue constructs were generated in a multi-well plate (A) or within a multi-well culture insert (B), and were optionally placed in a suitable multi-well plate for long-term maintenance and maturation. Here, tissue constructs were bioprinted in a 48-well polystyrene plate (A) and on a multi-porous membrane in a 6-well culture insert (B).

Figure 17 is a non-limiting example of a bioprinted hepatic tubular structure designed and printed for in vitro support. A 40 mm long bio-ink cylinder containing 70% HepG2/25% HUVEC/5% hepatic stellate cells was bioprinted and conditioned. A channel was created in the center of the structure using a removable bio-insert hydrogel.

Figure 18 is a series of non-limiting examples of planar and layered geometries (including combinations thereof) that are compatible with the construction methods described herein and that reproduce the structural or spatial elements and biology of native tissue structures. Exemplary geometries include structurally correct tissues with vascular networks (A), banded tissues (B), lobed tissues (C), perfused/aligned tissues (D), tissues with solid+liquid/liquid interfaces (E), barrier tissues (F), and stratified tissues with layered geometries (G).

19 is a non-limiting illustration of an extracorporeal liver device containing repeating tubular structures with evenly spaced void spaces (depicted as occupied by temporary filler bodies; dark circles) allowing perfusion across and/or through the engineered liver tissue.

FIG. 20 is a non-limiting example of bioprinted human liver tissue.

FIG21 is a series of non-limiting micrographs showing histological characterization of bioprinted human liver tissue; in this case, the micrographs illustrate high tissue density (A), production of ECM (B), production of lipids (C), and production of glycogen (D).

22 is a series of non-limiting micrographs showing histological characterization of bioprinted human liver tissue; in this case, the micrographs illustrate regions of hepatocytes surrounded by non-parenchymal stroma and apparent self-organization into lobular structures that mimic native liver tissue.

FIG23 is a non-limiting example of data showing albumin (A) and cholesterol (B) production from bioprinted human liver tissue.

FIG24 is a non-limiting example of data showing transferrin (A) and fibrinogen (B) production from bioprinted human liver tissue.

25 is a non-limiting example of data showing the relative amounts of bile salt exporter mRNA relative to housekeeping gene RNA (18s RNA) in bioprinted human liver tissue at day 10 after fabrication.

26 is a non-limiting example of data showing sustained induction of active CYP3A4 enzyme by rifampicin treatment in bioprinted human liver tissue (A), which can be inhibited by verapamil (B).

27 is a non-limiting example of data showing induction of CYP3A4 in bioprinted liver tissue by diclofenac treatment followed by tissue injury at higher doses as measured by lactate dehydrogenase (LDH).

28 is a non-limiting example of data showing albumin reduction (A) and LDH increase (B) in a multiple dose study of trovafloxacin versus levofloxacin in bioprinted liver tissue.

29 is a non-limiting example of data showing decreased viability (A), decreased albumin (B), and increased LDH (C) as measured by ATP in a multiple dose study of methotrexate in bioprinted liver tissue.

FIG30 is a non-limiting example of data showing the response of bioprinted liver tissue to Vitamin D as measured by induction of CYPs.

FIG31 is a non-limiting example of data showing the response to LPS stimulation in bioprinted liver tissue containing Kupffer cells as measured by IL-6 production.

Figure 32 is a non-limiting example of data showing the dose-dependent response to LPS (10 μg/ml (A) and 100 μg/ml (B)) stimulation in bioprinted liver tissue containing Kupffer cells as measured by cytokine (IL-10, IL-1b, and TNF-α) production.

Figure 33 is a non-limiting example of a hepatic sinusoidal endothelial cell (HSEC) monolayer that is approximately 90% confluent.

FIG34 is a non-limiting example of data showing the response of HSECs to LPS in two-dimensional culture as measured by IL-6 production.

FIG35 is a non-limiting example of a three-dimensional bioprinted liver tissue incorporating confluence of HSECs in the non-parenchymal portion.

FIG36 is a non-limiting example of a photomicrograph (FITC; 5X) of three-dimensional bioprinted liver tissue depicting small molecule penetration through a cryosection.

DETAILED DESCRIPTION

For many patients with liver failure, restoring even a portion of their liver mass by delivering healthy cells or tissue could have a significant impact on the patient's survival and liver function. According to the 2011 UNOS database, there are over 10,000 patients in the United States waiting for a liver transplant, of which less than 50% actually receive a transplanted liver. Many more patients could benefit from a transplanted liver than are listed on the transplant list, which is typically reserved for the sickest patients. Moreover, many individuals suffer from chronic degenerative diseases for which transplantation is not a viable option in today's healthcare model. Therefore, functional liver tissue would have enormous clinical value. In the United States, segmental or partial liver transplants are now routinely performed, in which a single organ is divided into 2-3 pieces or lobes, each of which is transplanted into a patient on the waiting list. Because healthy liver tissue has the unique ability to grow and adapt to an individual's size and metabolic needs, the approach of partial-mass transplantation is clinically feasible.

Furthermore, many compounds fail in late-stage clinical trials or post-marketing due to unexpected toxicities, which account for 35% of compound failures in Phase III (Curr. Pharm. Des. 2005 11:3545). Unexpected hepatotoxicity accounts for the majority of these failures, followed by cardiotoxicity and nephrotoxicity. Therefore, better systems are needed to provide more accurate predictions of in vivo performance in humans.

There is a need for materials, tools, and techniques that can greatly increase the number and quality of innovative, cost-effective new drugs without incurring prohibitive research and development costs. Accordingly, the inventors herein disclose engineered liver tissue constructs, arrays thereof, and methods for their preparation, which have utility in tissue and organ engineering, in vitro analysis, drug discovery, and other fields.

The previous model of liver tissue focuses on by being seeded into preformed and being shaped as the three-dimensional scaffold material that adapts to expected application, or makes hepatocyte form multicellular spheroid (wherein produced cell spheroid aggregate but there is no pointing structure in aggregate) by random assembly, provides the tissue construct of through engineering approaches.The cell that has been seeded on the scaffold material is primary cell, cell line, engineered cell and/or stem cell/progenitor cell.When utilizing pluripotent stem cell or progenitor cell, they or experience differentiation program in two-dimensional monolayer culture before being seeded on the three-dimensional scaffold material, or first be seeded on the scaffold material and subsequently in situ or experience differentiation program in vitro to produce desired tissue.With regard to the overall cellularity and structure of the required time of terminal differentiation of cell in cell yield, construct body and gained three-dimensional structure, traditional method is both laborious and inefficient.

Importantly, previous efforts and technology fail to produce the tissue compartments of restriction, and this compartment has specific cell types in the specific position in the structure and once keeps several days or weeks (up to 40 days) in construct. In addition, previous efforts and technology fail to show the natural sample structure that helps tissue life span, including microvascular structure and tight junction. Technology as herein described enables structure to be finely controlled and enables the geometry of highly restricted similar native tissue to be produced. In addition, this technology enables the high cell density tissue that keeps these geometries to be built, therefore prolongs the service life of tissue.

In certain embodiments, disclosed herein are engineered, living, three-dimensional liver tissue constructs comprising at least one compartment defining a planar geometry, the compartment comprising an interior bounded by sides, the interior comprising parenchymal cells, the sides comprising non-parenchymal cells; the cells coagulating to form a living, three-dimensional liver tissue construct; provided that at least a portion of the construct is bioprinted and the construct is substantially free of a preformed scaffold when in use.

Also disclosed herein, in certain embodiments, is an engineered, living, three-dimensional liver tissue construct comprising: one or more layers, each layer comprising one or more liver cell types, the one or more layers cohesing to form a living, three-dimensional liver tissue construct, the construct being characterized by having at least one of the following: at least one layer comprising a plurality of cell types spatially arranged relative to each other to create a planar geometry; and a plurality of layers, at least one layer being compositionally or structurally different from at least one other layer to create a laminar geometry.

Also disclosed herein, in certain embodiments, are engineered, living, three-dimensional liver tissue constructs comprising: a plurality of layers, each layer comprising a cylindrical bio-ink, the bio-ink being aligned with substantially parallel axes, the bio-ink comprising parenchymal hepatocytes; and optionally, non-parenchymal hepatocytes within or between the cylindrical bio-inks; and optionally, void spaces between the cylindrical bio-inks.

Also disclosed herein, in certain embodiments, are arrays of engineered, living, three-dimensional liver tissue constructs, each construct comprising: one or more layers, each layer comprising one or more liver cell types, the one or more layers cohesing to form a living, three-dimensional liver tissue construct, each construct being characterized by having at least one of the following: at least one layer comprising a plurality of cell types spatially arranged relative to each other to create a planar geometry; and a plurality of layers, at least one layer being compositionally or structurally different from at least one other layer to create a laminar geometry.

Also disclosed herein, in certain embodiments, are arrays of engineered, living, three-dimensional tissue constructs, wherein at least one of the constructs is a liver tissue construct, each liver tissue construct comprising: one or more layers, each layer comprising one or more liver cell types, the one or more layers cohesing to form a living, three-dimensional liver tissue construct, the liver tissue construct being characterized by having at least one of the following: at least one layer comprising a plurality of cell types spatially arranged relative to each other to create a planar geometry; and a plurality of layers, at least one layer being compositionally or structurally different from at least one other layer to create a laminar geometry.

In certain embodiments, the present invention also discloses a method for making a living three-dimensional liver tissue construct, comprising: preparing one or more bio-inks comprising non-parenchymal cells; preparing one or more bio-inks comprising parenchymal cells; depositing the bio-inks onto a support; and incubating the deposited bio-inks for about 1 hour to about 30 days to form a living three-dimensional liver tissue construct comprising at least one compartment comprising an interior comprising parenchymal cells bounded by a side comprising non-parenchymal cells.

In certain embodiments, the present invention also discloses a method for making 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 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 coagulating to form a living three-dimensional liver tissue construct.

In certain embodiments, also disclosed herein is a method for constructing a living three-dimensional liver tissue construct, the method comprising: preparing one or more cohesive multicellular aggregates comprising mammalian liver cells; placing the one or more cohesive multicellular aggregates on a support to form at least one of: at least one layer comprising a plurality of cell types spatially arranged relative to each other to create a planar geometry; and a plurality of layers, at least one layer being compositionally or structurally different from at least one other layer to create a laminar geometry; and incubating the one or more multicellular aggregates for about 1 hour to about 30 days to allow the multicellular aggregates to cohere and form a living three-dimensional tissue construct.

Also disclosed herein, in certain embodiments, is a method for making a living three-dimensional liver tissue construct, comprising incubating one or more bio-inks comprising parenchymal cells and one or more bio-inks comprising non-parenchymal cells deposited onto a support for about 1 hour to about 30 days to form a living three-dimensional liver tissue construct, the construct comprising at least one compartment comprising an interior comprising parenchymal cells bounded by a side comprising non-parenchymal cells.

In certain embodiments, the present invention also discloses an engineered, living, three-dimensional liver tissue construct comprising one or more layers, at least one layer comprising at least two cell types, the at least two cell types comprising a parenchymal liver cell type and a non-parenchymal liver cell type, the at least two cell types spatially arranged relative to each other to create a planar geometry comprising at least one parenchymal liver cell compartment and at least one non-parenchymal liver cell compartment, the one or more layers cohesing to form a living, three-dimensional liver tissue construct, provided that the living, three-dimensional liver tissue construct remains substantially viable in in vitro culture for at least 7 days and possesses at least two of the following liver characteristics: synthesis of cholesterol; storage of lipids; storage of glycogen; expression of liver-specific proteins comprising two or more of the following: albumin, fibrinogen, transferrin, CYP450 , alpha-1-antitrypsin, ornithine transcarbamylase, glycogen synthase, factor VIII and factor IX; responsive to hepatomodulators, including one or more of the following: hormones, inducers, toxins, drugs, antibodies, interfering RNA, small RNA, locked nucleic acid, viruses, bacteria, parasites and inflammatory inducers; responsive to hepatotoxicants, including one or more of the following: acetaminophen, alcohol, trovafloxacin, methotrexate, diclofenac, troglitazone, valproic acid, amiodarone; express transporters, including one or more of the following: BSEP, OATP1B1, NTCP, PGP, BCRP, OATP1B3, ABCB4, ATP8B1, multidrug resistance transporter (MDR); maintain at least some quiescent astrocytes after manufacturing; and contain microvascular structures.

Certain definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a nucleic acid" includes one or more nucleic acids, and/or compositions of the type described herein, as will be apparent to those skilled in the art upon reading this disclosure and so forth. Any reference herein to "or" is intended to include "and/or" unless otherwise indicated.

As used herein, "array" refers to a scientific tool comprising an association of multiple spatially arranged elements to allow multiple assays to be performed on one sample, one or more assays to be performed on multiple samples, or both.

As used herein, "assay" refers to a procedure for testing or measuring the presence or activity of a substance (e.g., a chemical, molecule, biochemical, protein, hormone, or drug, etc.) in an organic or biological sample (e.g., a cell aggregate, tissue, organ, organism, etc.).

As used herein, "biocompatible" means having a limited risk of causing damage or toxicity to cells. As presented in the specification and claims, "biocompatible porous containers" and "biocompatible membranes" have a limited risk of causing damage or toxicity to mammalian cells, but this definition does not extend to imply that these biocompatible elements can be implanted in mammals.

As used herein, "bio-ink" refers to a liquid, semi-solid, or solid composition used in bioprinting. In some embodiments, the bio-ink comprises a cell solution, a cell aggregate, a gel comprising cells, a multicellular body, or a tissue. In some embodiments, the bio-ink further comprises a support material. In some embodiments, the bio-ink further comprises a cell-free material that provides specific biomechanical properties that enable bioprinting. In some embodiments, the bio-ink comprises an extrusion compound.

As used herein, "bioprinting" refers to the three-dimensional precise deposition of cells (e.g., cell solutions, cell-containing gels, cell suspensions, cell concentrates, multicellular aggregates, multicellular bodies, etc.) via methods compatible with automated or semi-automated, computer-assisted three-dimensional prototyping devices (e.g., bioprinters).

As used herein, "cohere," "cohered," and "cohesion" refer to the property of cell-cell adhesion that binds cells, cell aggregates, multicellular aggregates, multicellular bodies, and/or layers thereof. The term can be used interchangeably with "fuse," "fused," and "fusion."

As used herein, "cylindrical" refers to having substantially the form of a cylinder. In various embodiments, the cylindrical bio-ink has substantially the form of a cylinder and is about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65% or 60% (including any amount therebetween) cylindrical in form. In further various embodiments, the cylindrical bio-ink has substantially the form of a cylinder along about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% or 50% (including any amount therebetween) of its length. In some embodiments, "cylindrical" refers to having substantially the form of a cylinder when manufactured.

As used herein, "lamellar" refers to a multilayered, bioprinted tissue in which two or more planar layers are combined to increase the total thickness of the tissue in the Z plane. In some embodiments, each planar layer is substantially similar in structure and/or composition. In other embodiments, each planar layer is substantially unique in structure and/or composition.

As used herein, a "layer" refers to a combination of cells that are multiple cell thicknesses in the X and Y planes. In some embodiments, the engineered liver tissue described herein comprises one layer. In other embodiments, the engineered liver tissue described herein comprises multiple layers. In various embodiments, the layers form a continuous, substantially continuous, or non-continuous sheet of cells. In some embodiments, each layer of the engineered liver tissue described herein comprises multiple cells in the X, Y, and Z axes. In further embodiments, each layer of the engineered liver tissue is at least about 50 microns thick in the X, Y, and Z axes.

As used herein, " multilayer " refers to be made up of two or more tissue layers, wherein each tissue layer is the thickness of one or more cell layers. In some embodiments, tissue layer is deposited one at a time. In other embodiments, multiple layers are deposited simultaneously. Optionally, each layer is made up of multiple cell types. Further, the multiple cell types in each layer are optionally arranged relative to each other in the X-Y plane (i.e.: horizontal plane) with the structure of spatial definition. In addition, in some cases, the layer increase on the Z- plane (i.e.: vertical plane) causes the controlled spatial positioning of cells relative to each other in the layer so that the structure of spatial definition continues on the Z- plane.

As used herein, "pluripotent cells" refer to cells that are capable of undergoing differentiation into two or more cell types. Pluripotent cells include, for example, mesenchymal stem cells/stromal cells, induced pluripotent stem cells, and embryonic stem cells.

As used herein, "parenchymal cells" refer to hepatocytes or hepatocyte-like cells; and "non-parenchymal cells" refer to liver cells that are not hepatocytes or hepatocyte-like cells.

As used herein, "planar" refers to a layer of bioprinted multicellular tissue in which multiple bio-ink compositions and/or void spaces are spatially arranged relative to each other in a defined pattern substantially within the X-Y plane of the tissue layer. See, for example, Figures 18A-E. In various embodiments, the planar layer is substantially planar, for example, about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, or 60% (including any amounts therebetween) are spatially arranged in a defined pattern within the X-Y plane of the tissue layer.

As used herein, "scaffold" refers to: synthetic scaffolds, such as polymer scaffolds and porous hydrogels; non-synthetic scaffolds, such as preformed extracellular matrix layers, dead cell layers, and decellularized tissues, as well as any other type of preformed scaffold that is essential to the physical structure of the engineered tissue and/or organ and cannot be removed from the tissue and/or organ without damaging/destroying the tissue and/or organ. In further embodiments, decellularized tissue scaffolds include decellularized natural tissue or decellularized cellular material produced by cultured cells in any manner; for example, cells that are caused to die or decellularize, leaving behind a layer of ECM that they produced when alive. Thus, the term "scaffoldless" is intended to imply that, when used, the scaffold is not an essential part of the engineered tissue, or has been removed or retained as an inert part of the engineered tissue. "Scaffoldless" can be used interchangeably with "scaffold-free" and "free of preformed scaffolds."

As used herein, "subject" refers to any individual that is a human, non-human animal, any mammal, or any vertebrate. The term is interchangeable with "patient," "recipient," and "donor."

As used herein, "support" refers to any biocompatible surface capable of accepting deposited bio-ink.

As used herein, "tissue" refers to an aggregate of cells. Examples of tissues include, but are not limited to, connective tissue (e.g., reticular connective tissue, dense connective tissue, elastic tissue, reticular connective tissue, and adipose tissue), muscle tissue (e.g., skeletal muscle, smooth muscle, and cardiac muscle), urogenital tissue, gastrointestinal tissue, lung tissue, bone tissue, neural tissue, and epithelial tissue (e.g., simple epithelium and stratified epithelium), tissue of endodermal origin, tissue of mesoderm origin, and tissue of ectoderm origin.

Tissue Engineering

Tissue engineering is an interdisciplinary field that applies and combines the principles of engineering and life sciences to develop biological substitutes that restore, maintain or improve tissue function by augmenting, repairing or replacing organs or tissues. The basic approach of classical tissue engineering is to seed living cells into a biocompatible and ultimately biodegradable environment (e.g., a scaffold), followed by culturing the construct in a bioreactor so that the initial cell population can further expand and mature to generate the target tissue after implantation. Using an appropriate scaffold that mimics the biological extracellular matrix (ECM), the developing tissue, in some cases, adopts the morphology and function of the desired organ after maturation in vitro and in vivo. However, due to the limited ability to control the distribution and spatial arrangement of cells throughout the scaffold, achieving a sufficiently high cell density with a structure similar to that of natural tissue is challenging. These limitations often result in tissues or organs with poor mechanical properties and/or insufficient function. Additional challenges exist in terms of the biodegradability of the scaffold, the inclusion of residual polymers, and the industrial scale-up of the manufacturing process. Scaffold-free approaches have been attempted. Current scaffold-free approaches are subject to several limitations:

Complex planar and/or layered geometries, such as multilayered structures where one or more layers differ compositionally or structurally from the other layers, or where one or more layers contain multiple cell types in spatially defined positions relative to each other, often require well-defined, high-resolution placement of cell types within a specific structure to reproducibly achieve results similar to native tissue.

• Size and/or geometry are limited by diffusion and/or nutrient supply requiring a functional vascular network.

• In some cases, tissue viability is compromised by confinement materials that restrict diffusion and limit cellular access to nutrients.

In certain embodiments, disclosed herein are engineered mammalian tissues, engineered liver tissues/constructs, arrays thereof, and methods of manufacture. The tissue engineering methods disclosed herein have the following advantages:

• They are capable of giving rise to tissues and/or organs comprising cells.

• By reproducing cell-cell interactions similar to those of native tissues, they mimic the environmental conditions found in the development, homeostasis and/or pathogenesis of native tissues.

• They optionally yield living three-dimensional tissues and composite tissues with a wide range of complex topologies and geometries (eg, multilayer structures, segments, sheets, tubes, cysts, etc.).

• They are compatible with automated or semi-automated manufacturing methods and are scalable.

Bioprinting enables improved methods for generating microscale tissue analogs, including those useful for in vitro analysis (see below).

Bioprinting

In some embodiments, at least a portion of the engineered liver tissue/construct and its array is bioprinted. In further embodiments, the bioprinted construct is obtained using a method using rapid prototyping technology, which is based on the three-dimensional, automated, computer-assisted deposition of cells and optional confining materials onto a biocompatible support surface (e.g., composed of a hydrogel and/or a porous membrane) by a three-dimensional delivery device (e.g., a bioprinter), wherein the cells include cell solutions, cell suspensions, gels or pastes containing cells, cell concentrates, multicellular bodies (e.g., cylinders, spheroids, ribbons, etc.). As used herein, in some embodiments, when used to refer to tissues and/or organs, the term "engineered" refers to: according to a computer script, by a computer-assisted device (e.g., a bioprinter), cells, cell solutions, cell suspensions, gels or pastes containing cells, cell concentrates, multicellular aggregates, and their layers are placed to form a three-dimensional structure. In further embodiments, the computer script is, for example, one or more computer programs, computer applications, or computer modules. In further embodiments, three-dimensional tissue structures are formed by post-printing fusion of cells or multicellular bodies, which in some cases resembles the self-assembly phenomenon in early morphogenesis.

While there are many methods that can be used to arrange cells, multicellular aggregates, and/or layers thereof on biocompatible surfaces to create three-dimensional structures, including manual placement, placement by automated, computer-assisted machines (e.g., bioprinters) is advantageous. Advantages of using this technology to deliver cells or multicellular bodies include: rapid, precise, and reproducible placement of cells or multicellular bodies to create constructs that exhibit planned or predetermined orientations or patterns of cells, multicellular aggregates, and/or layers thereof having various compositions. Advantages also include ensuring high cell density while minimizing cell damage. Parenchymal cells of the liver are particularly susceptible to damage by shear forces and other biomechanical stresses; therefore, the combined use of bioinks and the bioprinting process described herein provides unique advantages over alternative technologies, as highlighted by the favorable parenchymal cell viability after bioprinting shown in Figure 9.

In some embodiments, the bioprinting method is continuous and/or substantially continuous. A non-limiting example of a continuous bioprinting method is dispensing bio-ink (e.g., cells, cells in combination with excipients or extrusion compounds, or aggregates of cells) from a bioprinter via a dispensing end (e.g., a syringe, capillary, etc.) connected to a bio-ink reservoir. In a further non-limiting embodiment, the continuous bioprinting method is dispensing bio-ink in a repeating pattern of functional units. In various embodiments, the repeating functional units have any suitable geometry, including, for example, circular, square, rectangular, triangular, polygonal, and irregular geometries, thereby producing one or more tissue layers having a planar geometry obtained by spatial patterning of unique bio-inks and/or interstitial spaces. In a further embodiment, a repeating pattern of bioprinted functional units comprises a layer, and multiple layers are bioprinted (e.g., stacked) adjacently to form an engineered tissue or organ having a layered geometry. In various embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more layers are bioprinted (e.g., stacked) adjacently to form an engineered tissue or organ. In further embodiments, one or more layers of tissue having a laminar geometry also have a planar geometry.

In some embodiments, the functional unit is composed of several fused or cohesive tubular structures that are arranged in the horizontal and vertical dimensions to form a continuous liver tissue structure containing evenly spaced void spaces or channels that enable diffusion across and/or through the engineered liver tissue. See Figures 17 and 19.

In some embodiments, the functional units of bioprinting are repeated in a mosaic pattern. A "mosaic pattern" is a graphic plane that fills the plane without overlap or gaps. Figure 6A shows an example of a functional unit that is optionally repeated to produce the mosaic pattern shown in Figures 6B-D and Figure 7. As non-limiting examples, advantages of continuous and/or mosaic bioprinting include increased productivity of bioprinted tissues. Another non-limiting exemplary advantage is that there is no need to align the bioprinter with previously deposited bioink elements. In some embodiments, continuous bioprinting facilitates printing larger tissues from large bioink reservoirs, optionally using a syringe mechanism. Continuous bioprinting is also a suitable method for printing spatially defined boundaries using extruded compounds, hydrogels, polymers, bioinks, or any printable material that can retain its shape after printing; wherein the created boundaries are optionally filled by bioprinting one or more bioinks, thereby creating mosaic tissues with spatially defined planar geometries, for example, see the embodiments illustrated in Figures 3A, 5A, 5B, 6, 8, and 11.

In some embodiments, the method of continuous bioprinting includes optimizing and/or balancing parameters such as print height, pump speed, robot speed, or a combination thereof, independently or relative to each other. In some cases, the bioprinter head speed for deposition is 3 mm/s, the dispensing height of the first layer is 0.5 mm, and the dispensing height of each subsequent layer increases by 0.4 mm. In some embodiments, the dispensing height is substantially equal to the diameter of the bioprinter dispensing tip. Without limitation, a suitable and/or optimal dispensing distance does not cause the material to flatten or adhere to the dispensing needle. In various embodiments, the bioprinter dispensing tip has an inner diameter of about 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 μm or more, including any amount therebetween. In various embodiments, the bio-ink reservoir of the bio-printer has a volume of about 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 cubic centimeters or more, including any amount therebetween. In some cases, when the residual pressure accumulation in the system is low, the pump speed is suitable or optimal. In some cases, a favorable pump speed depends on the ratio between the cross-sectional area of the reservoir and the distribution needle, and the larger the ratio, the lower the pump speed is needed. In some embodiments, suitable and/or optimal printing speed makes it possible to deposit uniform lines without affecting the mechanical integrity of the material.

Invention disclosed herein includes business method.In some embodiments, the speed and scalability of technology and method disclosed herein are used to design, build and operate for producing the engineered liver tissue and/or organ for implantation or for generating the industry and/or commercial facilities of the instrument (for research and development, such as in vitro analysis) based on cell.In further embodiments, engineered liver tissue and/or organ and array thereof are produced, stored, distributed, listed, promoted and sold, such as as cell array (such as microarray or chip), tissue array (such as microarray or chip) and test kit for bioanalysis and high-throughput drug screening.In other embodiments, engineered liver tissue and/or organ and array thereof are manufactured and used for carrying out bioanalysis and/or drug screening as service.

Engineered liver tissue

With reference to Figure 1, native liver tissue is composed of a variety of cell types, which are arranged in a unique, spatially patterned structure relative to each other. Each hepatic lobule constitutes a working functional unit of the liver and is roughly hexagonal in shape. Parenchymal cells, or hepatocytes, primarily constitute the lobule, while a variety of non-parenchymal cells (cholangiocytes, sinusoidal endothelial cells, astrocytes, and Kupffer cells) in an accessory position surround the lobule and emanate between the portal vein and the bile duct (along the periphery of the lobule) toward the central vein (in the center of the lobule). The positioning of the non-parenchymal cells and the flow of blood and bile along the sinusoids and bile ducts, respectively, act together to form a suitable microenvironment for hepatocytes, and therefore affect the function and health of those cells. Various pathological conditions, such as cirrhosis or fibrosis in the liver, lead to the loss of key metabolic functions because the hepatocyte microenvironment is destroyed and the liver structure is progressively destroyed due to the excessive production of extracellular matrix, inflammation, and the production of pathogenic cytokines and growth factors.

Bioprinting enables the three-dimensional generation of compartmentalized liver tissue from a variety of cell inputs, thereby reproducing key elements of native tissue structure and function. Lobulated tissues can be generated according to the examples provided herein, in which parenchymal and non-parenchymal cells are spatially arranged relative to each other, thereby generating a planar geometry within each layer of tissue fabricated. Subsequently, layers can be added that precisely replicate the geometry of the first layer, or introduce additional features such as void spaces or channels or additional biological elements such as tumor cells or other biological or biochemical components associated with pathology or repair/regeneration processes. In addition to lobulated patterns, one can appreciate using the accompanying drawings that bioprinted tissues can be generated that replicate specific spatial relationships within a tissue, including but not limited to: blood vessels/parenchymal cells; blood vessels/hepatocytes; hepatocytes/bile ducts; fibrotic tissue/vasculature; fibrotic tissue/hepatocytes; hepatocytes/immune cells; parenchymal cells/non-parenchymal cells, liver sinusoids/blood or blood substitutes. In further embodiments, the liver tissue analog comprises: hepatocytes or hepatocyte-like cells and optionally bile duct epithelial cells and optionally non-parenchymal cell types including, but not limited to, astrocytes, endothelial cells, Kupffer cells, immune cells, or myofibroblasts.

In certain embodiments, disclosed herein are engineered liver tissues comprising coagulated mammalian cells and further comprising one or more layers of mammalian cells, wherein at least a portion of the tissue is bioprinted. In certain embodiments, one or more of the tissue layers are characterized by a planar geometry, wherein multiple cell types or bioink types and/or void spaces are present in spatially defined locations in the X-Y plane. In some embodiments, the tissue is multilayered, wherein at least one layer differs structurally or compositionally from the other layers, imparting a specific layered geometry to the tissue. In further embodiments, the layers have similar thicknesses in the Z plane. In still further embodiments, the layers have variable thicknesses in the Z plane. In further embodiments, any single layer is one cell layer thick. In some embodiments, the tissue is a liver analog. In further embodiments, the liver tissue analog comprises: hepatocytes or hepatocyte-like cells, and optionally bile duct epithelial cells, and optionally non-parenchymal cell types, including but not limited to: astrocytes, endothelial cells, Kupffer cells, immune cells, or myofibroblasts. In some embodiments, the resulting liver tissue construct has a thickness in the X, Y, and Z planes of at least about 50 microns.

In some embodiments, the engineered liver tissue/construct is bioprinted, which is a method described herein. In further embodiments, at least a portion of the engineered tissue is bioprinted. In further embodiments, another portion of the tissue is bioprinted. In some embodiments, as further described herein, when manufactured or used, the tissue does not contain any preformed scaffold. In some embodiments, as a result of being manufactured by tissue engineering techniques (including bioprinting), as part of an organism, the tissue of the present invention is further distinguished from tissue developed in vivo.

In some embodiments, the engineered liver tissue comprises any type of mammalian cells. In various further embodiments, the engineered liver tissue comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more cell types. In some embodiments, the cells of the engineered liver tissue are "cohesive" or "adherent" to each other. In further embodiments, cohesion or adhesion refers to the cell-cell adhesion properties of bound cells, multicellular aggregates, multicellular bodies and/or layers thereof.

In some embodiments, the engineered liver tissue/construct comprises one or more cell layers. In further embodiments, one or more layers in the layer are characterized by having a planar geometry. In further embodiments, multiple layers of the engineered tissue have a planar geometry; wherein the planar geometry is variable or the same between layers. In further embodiments, the planar geometry (X-Y plane) in the single layer is arranged in the Z-plane during manufacturing to create an additional geometry in the Z-plane of the composite tissue. In some embodiments, one or more layers within the multilayer structure are further characterized by having a planar geometry.

In some embodiments, the cell layer comprises one or more cell sheets. In various embodiments, the cell sheet is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more cells thick (including any amount therebetween). In other various embodiments, the cell sheet is about 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 μ m thick or thicker (including any amount therebetween). In some embodiments, the tissue layer comprises fused cell aggregates. In further embodiments, prior to fusion, the cell aggregates have a defined shape and/or structure, by way of non-limiting example, substantially spherical, elongated, substantially cylindrical, and ribbon-like shapes. In various embodiments, the fused cell aggregates form a layer that is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 μm thick or more, including any amount therebetween.

In further embodiments, the one or more layers comprise mammalian cells of any type. In various further embodiments, each layer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more cell types. In some embodiments, the engineered liver tissue/construct comprises one or more layers of endothelial cells on one or more surfaces. In other embodiments, the engineered liver tissue/construct comprises one or more layers of epithelial cells on one or more surfaces.

In various embodiments, the engineered liver tissue is of any suitable size. In some embodiments, the size of the bioprinted liver tissue changes over time. In further embodiments, the engineered liver tissue shrinks or shrinks after bioprinting due to, for example, cell migration, cell death, cell-cell interactions, compression, or other forms of shrinkage. In other embodiments, the engineered liver tissue grows or expands after bioprinting due to, for example, cell migration, cell growth and proliferation, production of extracellular matrix or other cell-producing portions of native tissue, maturation of cells/tissue, or other forms of expansion.

In some embodiments, the physical size of the engineered liver tissue is limited by the ability of nutrients, including oxygen, to diffuse into the interior of the construct. In various embodiments, the engineered liver tissue/construct is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 30 ... , 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 μm. In various embodiments, the engineered liver tissue is at least about 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, or 5.0 mm in its smallest dimension when bioprinted. In further embodiments, the engineered liver tissue/construct is about 25 μm to about 500 μm in its smallest dimension when bioprinted.

In various embodiments, the engineered liver tissue is of any suitable shape. In some embodiments, the shape is selected to mimic a specific native tissue or organ. In further embodiments, the shape is selected to mimic a specific pathology, condition, or disease state. In some embodiments, the engineered liver tissue has a substantially planar shape. In further embodiments, the planar tissue has any suitable planar geometry, including, by way of non-limiting example, a square, rectangle, polygon, circle, oval, or irregular shape. In some embodiments, the planar geometry is formed in the engineered liver tissue by the positioning of specific cells or bio-ink portions and/or void spaces relative to each other in the X-Y plane.

In some embodiments, the engineered liver tissue/construct is secured within the containment vessel in a manner suitable for spatially securing the position of the tissue relative to the containment vessel. In some embodiments, the engineered liver tissue is confined by a biocompatible material that provides physical support on one or more sides and limits the space occupied by the engineered liver tissue. In further embodiments, the engineered liver tissue is secured to a surface. In further embodiments, the engineered liver tissue is secured to a biocompatible surface. In yet further embodiments, multiple tissues are associated to form an array by being secured to a surface and spatially arranged, as described herein. In some embodiments, the engineered liver tissue is subjected to shear forces caused by fluid flow on one or more sides. In further embodiments, the applied shear forces are used to promote tissue maturation and development and/or promote migration, differentiation, proliferation, or deposition of extracellular matrix; or to promote transport of proteins or molecules into or out of cells within the tissue. In other embodiments, the engineered liver tissue undergoes continuous or periodic perfusion, recirculation, or agitation of liquid nutrients on one or more surfaces. In other embodiments, the engineered liver tissue and arrays thereof are housed in a multi-well bioreactor that provides continuous or periodic recirculation of liquid culture medium for each construct.

Tissue geometry

Natural tissue is characterized by the presence of spatial and compositional patterns driven by the tissue's cellular and extracellular (i.e., void space, extracellular matrix, proteinaceous material, etc.) components. An inherent challenge in tissue engineering strategies that deploy synthetic scaffolds to achieve three-dimensional shapes is the inability to replicate both the geometry and biological properties of natural tissue. To date, attempts to construct laminar or planar geometries resembling natural tissue within scaffold structures, while also allowing for the introduction of cells at densities mimicking natural tissue, have been hampered by technological limitations. Bioprinting overcomes both of these inherent challenges (planar/laminar geometry and cell density) through the spatially confined deposition of bio-inks composed of cells, as exemplified in Figures 18A-E. In some embodiments, planar geometries are created from multiple bio-ink formulations, thereby fabricating two or more tissue components (i.e., mesenchymal, epithelial, vascular, skeletal, cartilaginous, parenchymal, cortical, myeloid, papillary, lobular, etc.) by placing each tissue component/cell population/bio-ink formulation in a constrained position relative to one another within the X, Y, and/or Z planes. In some embodiments, this plane geometry introduces void space.In further embodiments, the void space in the plane geometry accommodates the fluid of at least one part of simulated body fluid, such as blood, lymph, bile, urine, secretions etc. In further embodiments, this void space optionally contains non-adhesion cell type or body fluid derived part (such as, hemocyte, bone marrow cell, lymphocyte, immune cell, cancer cell, platelet, protein etc.). In further embodiments, the non-adhesion cell type of body fluid derived part optionally exists as non-void space part, and this non-void space part has been introduced into the part comprising cell of this plane geometry before, during or after manufacturing. In further embodiments, non-adhesion cell part or body fluid derived part replenish the space that comprises cell in entering the plane geometry from void space, as the result of cell-cell interaction or to secretory factor reaction.

In some embodiments, fluid flow or perfusion is optionally initiated through the void space within the geometric shape. In some embodiments, the planar geometry allows for the creation of a tissue-tissue or tissue-fluid interface. In further embodiments, the tissue is fabricated in a container that is optically transparent to allow for real-time observation of cells at the interface created by the geometric shape.

In some embodiments, the tissue comprises a plurality of layers, wherein at least one of these layers is different in structure or composition from the other layers in the construct, thereby creating a laminar structure in the Z-plane. Examples of laminar structures include barrier tissues having an endothelial or epithelial barrier to the underlying interstitial tissue, as depicted by the example shown in Figure 18F. In some embodiments, one or more layers of tissue incorporate vascular or microvascular portions. In further embodiments, the merging of vascular or microvascular portions results in the formation of microvessels or pseudovascular networks in one or more portions of the engineered tissue. In some embodiments, one or more portions of the tissue having a laminar geometry are bioprinted. In some embodiments, one or more tissues having a laminar geometry are manufactured adjacent to each other, thereby producing a tissue interface.

In some embodiments, according to the non-limiting example illustrated in FIG18G , one or more layers of a multilayered engineered tissue having a laminar geometry also include a planar geometry. In some embodiments, the same planar geometry is continued in each layer, resulting in a three-dimensional tissue having a continuous structure in the X, Y, and Z planes. In some embodiments, the composition or planar geometry of one or more laminar layers is varied, such that the resulting three-dimensional tissue has a complex structure in all of the X, Y, and Z planes.

Referring to FIG18A , in a specific embodiment, an engineered liver tissue is fabricated having a planar geometry that represents a structurally correct tissue with a vascular network. In this embodiment, the engineered liver tissue is fabricated using a first bio-ink comprising parenchymal cells, such as hepatocytes. Each layer of parenchymal cells optionally comprises any planar pattern. In this embodiment, the engineered liver tissue is fabricated using a second bio-ink comprising vascular-associated cells, such as endothelial cells, smooth muscle cells, and fibroblasts, to form a vascular network. Optionally, this structure can be created by first bioprinting and establishing a vascular network, and then, in a second step, fabricating liver tissue around the vascular network.

18B , in a specific embodiment, engineered liver tissue is fabricated with a planar geometry that represents the tissue's ribbon-like connections. For example, this can be used to replicate three specific regions of the hepatic acinus from the periportal region to the central lobular region.

Referring to FIG18C , in a specific embodiment, engineered liver tissue is fabricated with a planar geometry representing lobulated tissue. In this embodiment, the engineered liver tissue is fabricated using a first bio-ink comprising hepatocytes to form lobules. Each geometric lobule optionally comprises spatially oriented structures within its boundaries. In this embodiment, the engineered liver tissue is fabricated using a second bio-ink comprising stromal/vascular cells to form a border around the lobules.

18D , in a specific embodiment, engineered liver tissues are produced having a planar geometry that represents perfused/arranged tissue. In this embodiment, the first component forms a channel, vessel, or tube with a lumen. In this embodiment, the second component forms a tissue patch that is optionally of the same size/shape or of different sizes/shapes, optionally comprises one or more cell types, and optionally comprises one or more spatial patterns achieved by directional patterning in the X, Y, and/or Z planes.

Referring to FIG18E , in a specific embodiment, engineered liver tissue is fabricated with a planar geometry representing a solid-liquid/liquid tissue interface. In this embodiment, a first bio-ink forms the outer wall of the luminal structure, a second bio-ink forms the inner wall of the luminal structure, and a third component is optionally a fluid containing cells or other biologically relevant components.

18F , in a specific embodiment, an engineered liver tissue is produced having a layered geometry that represents a barrier tissue. In this embodiment, the barrier layer is optionally endothelial or epithelial cells and the interstitial layer forms the wall and/or surface of the luminal tissue, which is seated on a porous mesh or membrane.

18G , in a specific embodiment, an engineered liver tissue having multiple layers is produced, each layer having a planar geometry and stacked to form a tissue having a layered geometry. In this embodiment, the planar geometry of the X and Y axes is continued into the produced tissue through the Z axis. The features of this geometry optionally include continuous channels or cell compartments.

cell

In some embodiments, disclosed herein is an engineered liver tissue (for example, liver analog) comprising one or more types of mammalian cells. In a further embodiment, the engineered liver tissue comprises hepatocytes or hepatocyte-like cells. In a further embodiment, suitable hepatocytes or hepatocyte-like cells include but are not limited to primary hepatocytes, cell lines such as HepG2 cells, tissue-specific progenitor cells such as HepaRG cells or a combination thereof. In a further embodiment, the engineered liver tissue comprises bile duct epithelial cells or bile duct epithelial-like cells. In a further embodiment, the engineered liver tissue comprises non-parenchymal cells. In a further embodiment, suitable non-parenchymal cells include but are not limited to vascular cells, endothelial cells, fibroblasts, myofibroblasts, adipocytes, adipogenic cells, mesenchymal cells, immune cells, Kupffer cells, astrocytes, bile duct epithelial cells, bile duct epithelial-like cells, sinusoidal endothelial cells, stem cells/progenitor cells derived from the liver, stem cells/progenitor cells not derived from the liver and a combination thereof.

In some embodiments, any mammalian cell is suitable for being included in engineered liver tissue and array thereof.In further embodiments, at least one part of this engineered liver tissue is an adhesion cell type.In further embodiments, for non-limiting example only, this mammalian cell is hepatocyte, parenchymal cell, non-parenchymal cell, contractility or muscle cell (for example, smooth muscle cell and myoblast), connective tissue cell (for example, fibroblast), bone marrow cell, endothelial cell, epithelial cell, vascular cell, blood cell, lymphocyte, pericyte, mesothelial cell, stromal cell, undifferentiated cell (for example, embryonic cell, stem cell and progenitor cell), cell derived from endoderm, cell derived from mesoderm, cell derived from ectoderm and combination thereof.

In some embodiments, the engineered liver tissue comprises endothelial cells, such as human endothelial cells. In some embodiments, suitable endothelial cells are derived from tissues including, by way of non-limiting example, liver tissue, blood, blood vessels, lymphatic vessels, digestive tract tissue, urogenital tract tissue, adipose tissue, respiratory tract tissue, reproductive system tissue, bone marrow, and umbilical cord tissue.

In some embodiments, the cell is an adult, differentiated cell. In a further embodiment, a "differentiated cell" is a cell having a tissue-specific phenotype consistent with, for example, a hepatocyte or endothelial cell when separated, wherein the tissue-specific phenotype (or the potential for displaying the phenotype) is maintained from the time of separation to the time of use. In other embodiments, the cell is an adult, undifferentiated cell. In a further embodiment, an "undifferentiated cell" is a cell that does not have or has lost a clear tissue-specific trait. In some embodiments, undifferentiated cells include stem cells. In a further embodiment, a "stem cell" is a cell that exhibits potential and self-renewal. Stem cells include, but are not limited to, totipotent stem cells, pluripotent stem cells, multipotent cells, oligopotent cells, unipotent cells, and progenitor cells. In various embodiments, stem cells are embryonic stem cells, adult stem cells, amniotic membrane stem cells, and induced pluripotent stem cells. In other embodiments, the cell is a mixture of adult differentiated cells and adult undifferentiated cells.

In some embodiments, the engineered liver tissue comprises parenchymal cells derived from pluripotent stem cells/progenitor cells. In various embodiments, the parenchymal cell source is suitably derived from fetal mammalian liver tissue; embryonic stem cells (ESC); induced pluripotent stem cells (iPSC); adult stem cells/progenitor cells derived from the liver; and adult stem cells/progenitor cells derived from non-liver tissue. In some embodiments, the engineered liver tissue is derived from pluripotent stem cells/progenitor cells that have been partially or completely differentiated into endoderm or liver phenotypes before use in the manufacture of liver tissue constructs.

In some embodiments, the construct comprises stem cells/progenitor cells that have been exposed to one or more differentiation signals. In further embodiments, the differentiation signals include one or more of the following: biomechanical signals, resolvable/soluble signals (e.g., biochemical signals) and physical signals. At many time points during the engineered liver tissue manufacturing process, stem cells/progenitor cells are suitably exposed to one or more differentiation signals. In some embodiments, the stem cells/progenitor cells are exposed to one or more differentiation signals before construct manufacture. In other embodiments, the stem cells/progenitor cells are exposed to one or more differentiation signals during construct manufacture. In other embodiments, the stem cells/progenitor cells are exposed to one or more differentiation signals after construct manufacture.

In various embodiments, the cell type and/or source of the cell is selected, configured, processed or adjusted based on a specific research purpose or goal. In some embodiments, one or more specific cell types are selected, configured, processed or adjusted to facilitate research on a specific disease or condition. In some embodiments, one or more specific cell types are selected, configured, processed or adjusted to facilitate research on a disease or condition in a specific subject. In some embodiments, one or more specific cell types are derived from two or more different human donors. In some embodiments, one or more specific cell types are derived from a specific vertebrate subject. In further embodiments, one or more specific cell types are derived from a specific mammalian subject. In further embodiments, one or more specific cell types are derived from a specific human subject. In further embodiments, one or more specific cell types are derived from a specific subject with a specific phenotype associated with a disease or tissue function. In further embodiments, subject-specific cells are separated from the target tissue of interest by means of a biopsy or tissue sampling. In further embodiments, subject-specific cells are used to manufacture tissues immediately after separation. In other embodiments, subject-specific cells are manipulated in vitro before being used to manufacture three-dimensional tissues; wherein the manipulation comprises one or more of the following: expansion, differentiation, directed differentiation, proliferation, exposure to proteins or nucleic acids, introduction of genetic vectors, introduction of genetic or non-genetic cell tracing parts, dedifferentiation (ie, generation of induced pluripotent stem cells or equivalents), cryopreservation. In some embodiments, subject-specific cells are separated from tissues other than the target tissue. In further embodiments, the subject-specific cells need to be differentiated into the cell type of interest within the target tissue. In yet further embodiments, the subject-specific cells that need to be differentiated are differentiated before, during, or after being manufactured into a three-dimensional structure.

Cell culture methods

The cell types used in the engineered liver tissue of the present invention are suitably cultured in any manner known in the art. Cell and tissue culture methods are known in the art and are described, for example, in Freshney, R., Culture of Animal Cells: A Manual of Basic Techniques, Wiley (1987), the contents of which are incorporated herein by reference for this information. Common mammalian cell culture techniques, cell lines, and cell culture systems suitable for use with the present invention are also described in Doyle, A., Griffiths, J.B., Newell, D.G., (eds.) Cell and Tissue Culture: Laboratory Procedures, Wiley (1998), the contents of which are incorporated herein by reference for this information.

The growth conditions that are suitable for the mammalian cells in culture are well known in the art. Cell culture medium generally includes essential nutrients and optional additional ingredients, such as growth factors, salts, minerals, vitamins, platelet-rich plasma etc., which are optionally selected according to the cell type cultivated. In some embodiments, specific composition is selected to enhance the secretion of cell growth, differentiation and specific proteins etc. Usually, standard growth medium includes Dulbecco's modified Eagle's medium (DMEM) containing 4g/L glucose, is supplemented with 1-20% fetal bovine serum (FBS), calf serum or human serum, insulin (4 micrograms/mL), dexamethasone (1.0 micromoles), amphotericin B (0.25 micrograms/mL) and Glutamax (1x). Williams E medium supplemented with insulin (1 g/L), sodium selenite (0.0067 g/L), transferrin (0.55 g/L), dexamethasone (1.0 micromolar), penicillin (100 U/mL), streptomycin (0.1 mg/mL), amphotericin B (0.25 micrograms/mL) and Glutamax (1x), or various other culture media known in the art. Preferably, the cells are cultured under sterile conditions in an atmosphere of 1-21% O ₂ and preferably 3-5% CO ₂ at a temperature close to or reaching the body temperature of the animal from which the cells were derived. For example, human cells are preferably cultured at about 37° C.

Cells are optionally cultured with cell differentiation agents to induce cells to differentiate along the desired route. For example, cells are optionally cultured with growth factors, cytokines, etc. In some embodiments, the term "growth factor" refers to a complex of proteins, polypeptides, or polypeptides, including cytokines, that are produced by cells and affect themselves and/or a variety of adjacent or distant cells. Generally speaking, growth factors affect the growth and/or differentiation of specific types of cells, either developmentally or in response to numerous physiological or environmental stimuli. Some, but not all, growth factors are hormones. Exemplary growth factors are insulin, insulin-like growth factor (IGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), fibroblast growth factors (FGFs, including basic FGF (bFGF)), platelet-derived growth factors (PDGFs, including PDGF-AA and PDGF-AB), hepatocyte growth factor (HGF), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β, including TGFβ1 and TGFβ3), epidermal growth factor (EGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin-6 (IL-6), IL-8, etc. Growth factors are discussed in, among other places, Molecular Cell Biology, Scientific American Books, Darnell et al., ed., 1986; Principles of Tissue Engineering, 2nd edition, Lanza et al., ed., Academic Press, 2000. Those skilled in the art will understand that any and all culture-derived growth factors in the conditioned media described herein are within the scope of the present invention.

Bioinks and multicellular aggregates

In certain embodiments, disclosed herein are three-dimensional living tissues (including liver tissue/constructs), arrays thereof, and methods thereof comprising bioprinted cells. In some embodiments, cells are bioprinted by depositing or extruding bio-ink from a bioprinter. In some embodiments, "bio-ink" comprises a liquid, semi-solid, or solid composition comprising a plurality of cells. In some embodiments, the bio-ink comprises a liquid or semi-solid cell solution, cell suspension, or cell concentrate. In further embodiments, the cell solution, suspension, or concentrate comprises a liquid or semi-solid (e.g., viscous) carrier and a plurality of cells. In further embodiments, the carrier is a suitable cell nutrient medium, such as those described herein. In some embodiments, prior to bioprinting, the bio-ink comprises a plurality of cells, which are optionally condensed into multicellular aggregates. In further embodiments, the bio-ink comprises a plurality of cells and is bioprinted to produce specific planar and/or layered geometries; wherein individual cells in the bio-ink condense before, during, and/or after bioprinting. In some embodiments, the bio-ink is produced by the following method: 1) collecting a plurality of cells in a fixed volume; wherein the cellular component accounts for at least about 30% and at most 100% of the total volume. In some embodiments, the bio-ink comprises semi-solid or solid multicellular aggregates or multicellular bodies. In a further embodiment, the bio-ink is produced by the following steps: 1) mixing a plurality of cells or cell aggregates with a biocompatible liquid or gel in a predetermined ratio to form a bio-ink; and 2) densifying the bio-ink to produce a bio-ink with a desired cell density and viscosity. In some embodiments, the densification of the bio-ink is achieved by centrifugation, tangential flow filtration ("TFF"), or a combination thereof. In some embodiments, the densification of the bio-ink produces an extrudable composition, thereby allowing the formation of multicellular aggregates or multicellular bodies. In some embodiments, "extrudable" means capable of being formed by being forced (e.g., under pressure) through a nozzle or orifice (e.g., one or more holes or tubes). In some embodiments, the densification of the bio-ink is caused by the cells growing to a suitable density. The cell density required for the bio-ink will vary depending on the cells used and the tissue or organ to be produced. In some embodiments, the cells of the bio-ink are cohesive and/or adherent. In some embodiments, "cohere," "cohered," and "cohesion" refer to the cell-cell adhesion property that binds cells, multicellular aggregates, multicellular bodies, and/or layers thereof together. In some embodiments, the term can be used interchangeably with "fuse," "fused," and "fusion." In some embodiments, the bio-ink further comprises a support material, a cell culture medium (or a supplement thereof), an extracellular matrix (or a component thereof), a cell adhesion agent, a cell death inhibitor, an anti-apoptotic agent, an antioxidant, an extrusion compound, and combinations thereof.

In various embodiments, the cell is any suitable cell. In further various embodiments, the cell is a vertebrate cell, a mammalian cell, a human cell, or a combination thereof. In some embodiments, the cell type used in the methods disclosed herein depends on the type of construct or tissue to be produced. In some embodiments, the bio-ink comprises one type of cell (also referred to as a "homogeneous" or "monotypic" bio-ink). In some embodiments, the bio-ink comprises more than one type of cell (also referred to as a "heterogeneous" or "polytypic" bio-ink).

Cell culture medium

In some embodiments, the bio-ink comprises a cell culture medium. The cell culture medium is any suitable culture medium. In various embodiments, suitable cell culture media include, by way of non-limiting example, Dulbecco's phosphate-buffered saline, Earle's balanced salts, Hanks' balanced salts, Tyrode's salts, Alsever's solution, Gey's balanced salt solution, Kreb's-Henseleit modified buffer system, Kreb's-Ringer bicarbonate buffer system, Puck's saline, Dulbecco's modified Eagle's medium, Dulbecco's modified Eagle's medium/Nutrient F-12 Ham, Nutrient Mixture F-10 Ham (Ham's F-10), Medium 199, Minimal Essential Medium Eagle, RPMI-1640 medium, Ames medium, BGJb medium (Fitton-Jackson modification), Click medium, CMRL-1066 medium, Fischer's medium, Glascow's Minimal Essential Medium (GMEM), Iscove's Modified Dulbecco's Medium (IMDM), L-15 medium (Leibovitz), McCoy 5A modified medium, NCTC medium, Swim S-77 medium, Waymouth medium, William's medium E, or a combination thereof. In some embodiments, the cell culture medium is modified or supplemented. In some embodiments, the cell culture medium further comprises albumin, selenium, transferrin, fetuin, sugars, amino acids, vitamins, growth factors, cytokines, hormones, antibiotics, lipids, lipid carriers, cyclodextrins, platelet-rich plasma, or a combination thereof.

Extracellular matrix

In some embodiments, the bio-ink further comprises one or more components of an extracellular matrix or a derivative thereof. In some embodiments, the "extracellular matrix" includes proteins produced by cells and transported from cells to the extracellular space, where they serve as supports to hold the tissue together, provide tensile strength and/or promote cell signaling. Examples of extracellular matrix components include, but are not limited to, collagen, fibronectin, laminin, hyaluronate, elastin, and proteoglycans. For example, in some embodiments, the multicellular aggregates contain a variety of ECM proteins (e.g., gelatin, fibrinogen, fibrin, collagen, fibronectin, laminin, elastin, and proteoglycans). Optionally, ECM components or derivatives of ECM components are added to the cell slurry for forming multicellular aggregates. The ECM components or derivatives of ECM components added to the cell slurry are optionally purified from human or animal sources, or produced by recombinant methods known in the art. In some embodiments, the derivative of ECM components or ECM components is secreted naturally by the cells in the elongated cell body, or the cell for forming the elongated cell body is optionally genetically manipulated by any suitable method known in the art, to change the expression level of one or more ECM components or derivatives of ECM components and/or one or more cell adhesion molecules or cell-substrate adhesion molecules (for example, selectins, integrins, immunoglobulins and adhesins). In some embodiments, the derivative of ECM components or ECM components promotes the cohesion of cells in multicellular aggregates. For example, gelatin and/or fibrinogen are suitably added to the cell slurry, and the slurry is used to form multicellular aggregates. By adding thrombin, fibrinogen is changed into fibrin.

In some embodiments, the bio-ink further comprises an agent that promotes cell adhesion.

In some embodiments, the bio-ink further comprises an agent that inhibits cell death (e.g., necrosis, apoptosis, or autophagy). In some embodiments, the bio-ink further comprises an anti-apoptotic agent. Agents that inhibit cell death include, but are not limited to, small molecules, antibodies, peptides, peptidomimetics, or combinations thereof. In some embodiments, agents that inhibit cell death are selected from: anti-TNF agents, agents that inhibit interleukin activity, agents that inhibit interferon activity, agents that inhibit GCSF (granulocyte colony-stimulating factor) activity, agents that inhibit macrophage inflammatory protein activity, agents that inhibit TGF-B (transforming growth factor B) activity (e.g., see Figures 15 and 16), agents that inhibit MMP (matrix metalloproteinase) activity, agents that inhibit caspase activity, agents that inhibit MAPK/JNK signaling cascade activity, agents that inhibit Src kinase activity, agents that inhibit JAK (Janus kinase) activity, or combinations thereof. In some embodiments, the bio-ink comprises an antioxidant. In some embodiments, the bio-ink comprises an oxygen carrier or other cell-specific nutrients.

Extrusion compounds

In some embodiments, the bio-ink further comprises an extrusion compound (i.e., a compound that alters the extrusion properties of the bio-ink). Examples of extrusion compounds include, but are not limited to, gels, hydrogels, peptide hydrogels, amino acid-based gels, surfactant polyols (e.g., Pluronic F-127 or PF-127), thermoresponsive polymers, hyaluronates, alginate, extracellular matrix components (and their derivatives), collagen, gelatin, other biocompatible natural or synthetic polymers, nanofibers, and self-assembled nanofibers. In some embodiments, after bioprinting, the extrusion compound is removed by physical, chemical, or enzymatic means.

Gels have been defined in various ways and are sometimes referred to as jelly. For example, the United States Pharmacopoeia defines gels as suspensions consisting of small inorganic particles or semisolid systems consisting of large organic molecules that are interpenetrated by a liquid. Gels include single-phase or two-phase systems. Single-phase gels are composed of organic macromolecules that are evenly distributed throughout the liquid in a manner that leaves no distinct boundary between the dispersed macromolecules and the liquid. Certain single-phase gels are prepared from synthetic macromolecules (e.g., carbomer) or from natural gums (e.g., tragacanth). In some embodiments, single-phase gels are typically aqueous, but can also be prepared using alcohols and oils. Two-phase gels consist of a network of small discrete particles.

In some cases, gels are classified as hydrophilic or hydrophobic. In certain embodiments, the matrix of a hydrophobic gel consists of liquid paraffin and polyethylene, or a fatty oil gelled with colloidal silica, or an aluminum or zinc soap. In contrast, the matrix of a hydrophilic gel generally consists of water, glycerol, or propylene glycol gelled with a suitable gelling agent (e.g., gum tragacanth, starch, cellulose derivatives, carboxyvinyl polymers, and magnesium aluminum silicate). In certain embodiments, the rheology of the compositions or devices disclosed herein is pseudoplastic, plastic, thixotropic, or dilatant.

Suitable hydrogels include those derived from collagen, hyaluronate, hyaluronic acid, fibrin, alginate, agarose, chitosan and combinations thereof. In other embodiments, suitable hydrogels are synthetic polymers. In further embodiments, suitable hydrogels include those derived from poly (acrylic acid) or derivatives thereof, poly (ethylene oxide) and copolymers thereof, poly (vinyl alcohol), polyphosphazene and combinations thereof. In each specific embodiment, limiting material is selected from: hydrogel, NovoGel ^™ , agarose, alginate, gelatin, Matrigel ^™ , hyaluronic acid, poloxamer, peptide hydrogel, poly (isopropyl n-polyacrylamide), polyethylene glycol diacrylate (PEG-DA), hydroxyethyl methacrylate, polydimethylsiloxane, polyacrylamide, poly (lactic acid), silicon, silk or combinations thereof.

In some embodiments, the extruded compound based on hydrogel is a thermoreversible gel (also referred to as thermoresponsive gel or thermogel). In some embodiments, suitable thermoreversible hydrogel is not liquid at room temperature. In a specific embodiment, the gelling temperature (Tgel) of suitable hydrogel is about 10 DEG C, 11 DEG C, 12 DEG C, 13 DEG C, 14 DEG C, 15 DEG C, 16 DEG C, 17 DEG C, 18 DEG C, 19 DEG C, 20 DEG C, 21 DEG C, 22 DEG C, 23 DEG C, 24 DEG C, 25 DEG C, 26 DEG C, 27 DEG C, 28 DEG C, 29 DEG C, 30 DEG C, 31 DEG C, 32 DEG C, 33 DEG C, 34 DEG C, 35 DEG C, 36 DEG C, 37 DEG C, 38 DEG C, 39 DEG C, 40 DEG C, including any amount therebetween. In certain embodiments, the Tgel of suitable hydrogel is about 10 DEG C to about 40 DEG C. In a further embodiment, the Tgel of suitable hydrogel is about 20 DEG C to about 30 DEG C. In some embodiments, the bio-ink described herein (e.g., comprising a hydrogel, one or more cell types and other additives, etc.) is not liquid at room temperature. In some embodiments, a suitable thermoreversible hydrogel is not liquid at mammalian body temperature. In a specific embodiment, the gelling temperature (Tgel) of a suitable hydrogel is about 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 41°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, including any amount therebetween. In certain embodiments, the Tgel of a suitable hydrogel is about 22°C to about 52°C. In a further embodiment, the Tgel of a suitable hydrogel is about 32°C to about 42°C. In some embodiments, the bio-ink described herein (e.g., comprising a hydrogel, one or more cell types, and other additives, etc.) is not liquid at mammalian body temperature. In specific embodiments, the gelling temperature (Tgel) of the bio-ink described herein is about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, including any amount therebetween. In specific embodiments, the Tgel of the bio-ink described herein is about 10°C to about 15°C. In another specific embodiment, the Tgel of the bio-ink described herein is about 15°C to about 20°C. In another specific embodiment, the Tgel of the bio-ink described herein is about 20°C to about 25°C. In another specific embodiment, the Tgel of the bio-ink described herein is about 25°C to about 30°C. In another specific embodiment, the Tgel of the bio-ink described herein is about 30°C to about 35°C. In another specific embodiment, the Tgel of the bio-ink described herein is about 35°C to about 40°C. In another specific embodiment, the bio-ink described herein has a Tgel of about 40°C to about 45°C. In another specific embodiment, the bio-ink described herein has a Tgel of about 45°C to about 50°C.

When incorporated into aqueous solutions, polymers composed of poly(propylene oxide) and polyethylene oxide form thermoreversible gels. These polymers have the ability to transition from a liquid to a gel at temperatures that can be maintained in bioprinter devices. This liquid-to-gel phase transition depends on the polymer concentration and the composition of the solution.

Poloxamer 407 (Pluronic F-127 or PF-127 or Lutrol) is a nonionic surfactant composed of a polyethylene oxide-polypropylene oxide copolymer. Other poloxamers include 188 (F-68 grade), 237 (F-87 grade), and 338 (F-108 grade). Aqueous solutions of poloxamers are stable in the presence of acids, bases, and metal ions. PF-127 is a commercially available polyethylene oxide-polypropylene oxide triblock copolymer of the general formula E106P70E106, with an average molar mass of 13,000. The polymer is optionally further purified using suitable methods to enhance the gelling properties of the polymer. It contains approximately 70% ethylene oxide, which explains its hydrophilicity. It is a member of the poloxamer ABA block copolymer series. PF-127 has good solubilizing power and low toxicity, and is therefore considered a suitable compound for extrusion.

In some embodiments, the viscosity of the hydrogels and bio-inks presented herein is measured by any of the described means. For example, in some embodiments, an LVDV-II+CP Cone Plate viscometer and a Cone Spindle CPE-40 are used to calculate the viscosity of the hydrogels and bio-inks. In other embodiments, a Brookfield (spindle and cup) viscometer is used to calculate the viscosity of the hydrogels and bio-inks. In some embodiments, the viscosity ranges mentioned herein are measured at room temperature. In other embodiments, the viscosity ranges mentioned herein are measured at body temperature (e.g., at the average body temperature of a healthy person).

In further embodiments, the hydrogel and/or bio-ink is characterized by having a viscosity of about 500 to 1,000,000 centipoise, about 750 to 1,000,000 centipoise, about 1000 to 1,000,000 centipoise, about 1000 to 400,000 centipoise, about 2000 to 100,000 centipoise, about 3000 to 50,000 centipoise, about 4000 to 25,000 centipoise, about 5000 to 20,000 centipoise, or about 6000 to 15,000 centipoise.

In some embodiments, the bio-ink comprises cells and an extrusion compound suitable for continuous bioprinting. In specific embodiments, the bio-ink has a viscosity of about 1500 mPa·s. In some embodiments, a mixture of Pluronic F-127 and cellular material is suitable for continuous bioprinting. Such a bio-ink is suitably prepared by dissolving Pluronic F-127 powder to 30% (w/v) by continuous mixing in cold (4°C) phosphate-buffered saline (PBS) for 48 hours. Pluronic F-127 is also suitably dissolved in water. In some embodiments, the cells are cultured and expanded using standard sterile cell culture techniques. In further embodiments, for example, the cells are pelleted at 200 g and resuspended in 30% Pluronic F-127 and aspirated into a reservoir attached to the bioprinter, which, in some embodiments, allows them to solidify at a gelling temperature of about 10°C to about 25°C. Gelation of the bio-ink prior to bioprinting is optional. Bio-inks, including bio-inks comprising Pluronic F-127, are optionally dispensed as a liquid.

In various embodiments, the concentration of Pluronic F-127 is any value that has a suitable viscosity and/or cytotoxicity. In some embodiments, the suitable concentration of Pluronic F-127 is capable of supporting weight while maintaining its shape during bioprinting. In some embodiments, the concentration of Pluronic F-127 is about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%. In some embodiments, the concentration of Pluronic F-127 is about 30% to about 40%, or about 30% to about 35%.

In some embodiments, the concentration of gelatin in the extrusion compound or excipient is 6% and the concentration of alginate is 0.5%. In other embodiments, the concentration of gelatin in the extrusion compound or excipient is 4% and the concentration of alginate is 2%.

In some embodiments, the non-cellular components of the bio-ink (e.g., extrusion compounds, etc.) are removed prior to use. In further embodiments, the non-cellular components are, for example, hydrogels, peptide hydrogels, amino acid-based gels, surfactant polyols, thermoresponsive polymers, hyaluronates, alginate, collagen, or other biocompatible natural or synthetic polymers. In further embodiments, the non-cellular components are removed by physical, chemical, or enzymatic means. In some embodiments, at the time of use, a portion of the non-cellular components remains associated with the cellular components.

In some embodiments, cells are pre-treated to increase cell interactions. For example, to enhance cell-cell interactions before forming the bio-ink, cells are suitably cultured in a centrifuge tube after centrifugation.

Exemplary cell ratios

In some embodiments, the bio-ink comprises multicellular bodies that further comprise non-parenchymal hepatocytes (e.g., endothelial cells, hepatic stellate cells, Kupffer cells). In further embodiments, the ratio of endothelial cells to hepatic stellate cells to Kupffer cells is any suitable ratio. In further embodiments, the ratio of endothelial cells to hepatic stellate cells to Kupffer cells is from about 90:10:0 to about 10:90:0. In still further embodiments, the ratio of endothelial cells to hepatic stellate cells to Kupffer cells is 45:45:10.

In some embodiments, the bio-ink comprises a multicellular body that further comprises hepatocytes (e.g., primary hepatocytes, HepG2, or HepaRG) and another cell type in a 100:0 ratio. In further embodiments, the ratio of hepatocytes to another non-parenchymal cell type (e.g., endothelial cells) is 95:5. In further embodiments, the ratio of stem cells to another non-parenchymal cell type (e.g., endothelial cells, astrocytes) is 50:50. In still further embodiments, the ratio of parenchymal cells to non-parenchymal cells (e.g., endothelial cells, astrocytes, Kupffer cells) is 50:35:10:5.

Cell self-sorting

In some embodiments, the multicellular aggregates used to form a construct or tissue include all cell types that would be included in the engineered tissue (e.g., hepatocytes, endothelial cells, hepatic stellate cells, and Kupffer cells); in such instances, each cell type migrates to a suitable location (e.g., during maturation) to form an engineered tissue, such as engineered liver tissue. In other embodiments, the multicellular aggregates used to form a structure include fewer cell types than all cell types that would be included in the engineered tissue. In some embodiments, each type of cell is evenly distributed in the multicellular aggregate, or in an area or layer of tissue. In other embodiments, each type of cell is localized in a specific area within the multicellular aggregate or in an area or layer of tissue.

For example, in the case of engineered liver tissue containing hepatocytes, endothelial cells, hepatic stellate cells, and Kupffer cells in appropriate ratios (e.g., 70:15:10:5), adjacent, bioprinted, cohesive multi-type bio-ink units fused together. During maturation, endothelial cells localized to some extent at the periphery and center of the construct and organized into microvascular structures. In some embodiments, the positioning of cell types within the construct mimics the hierarchical structure of mammalian tissue in vivo or ex vivo.

Preformed stents

In some embodiments, disclosed herein are engineered implantable tissues and organs that are free of or substantially free of any preformed scaffold. In further embodiments, "scaffold" refers to synthetic scaffolds, such as polymer scaffolds and porous hydrogels; non-synthetic scaffolds, such as preformed extracellular matrix layers, dead cell layers, and decellularized tissues; and any other type of preformed scaffold that is essential to the physical structure of the engineered tissue and/or organ and is not removed from the tissue and/or organ. In further embodiments, decellularized tissue scaffolds include decellularized natural tissue or decellularized cellular material produced by cultured cells in any manner; for example, a cell layer that is caused to die or decellularize, leaving behind the ECM that they produced when alive.

In some embodiments, the engineered liver tissue/construct and array thereof do not utilize any preformed scaffold, for example, to form any layer of the tissue, the tissue or the shape of the tissue. As non-limiting examples, when manufacturing or using, the engineered liver tissue of the present invention does not utilize any preformed synthetic scaffold (for example, polymeric scaffold), preformed extracellular matrix layer or any other type of preformed scaffold. In some embodiments, the engineered liver tissue is substantially free of any preformed scaffold. In further embodiments, the cellular component of the tissue contains detectable but trace or micro-amount of scaffold, for example, less than 2.0% of the total composition, less than 1.0% or less than 0.5% scaffold. In further embodiments, trace or micro-amount of scaffold is not enough to affect the long-term behavior of the tissue or its array or hinders its primary biological function. In other embodiments, after printing, scaffold components are removed by physical, chemical or enzymatic methods, thereby producing an engineered tissue that does not contain or is substantially free of scaffold components.

In some embodiments, the engineered liver tissue disclosed herein is free of or substantially free of preformed scaffolds, in stark contrast to those developed using certain other tissue engineering methods, in which a scaffold material is first formed, cells are then seeded onto the scaffold, and the cells subsequently proliferate to fill and assume the shape of the scaffold. In one aspect, the bioprinting methods described herein allow for the production of living and useful tissues that are free of or substantially free of preformed scaffolds. On the other hand, in some embodiments, a confinement material is utilized to maintain the cells of the present invention in a desired three-dimensional shape. The confinement material is different from a scaffold at least in that it is temporary and/or removable from the cells and/or tissue.

Array

In some embodiments, the array of engineered liver tissue/construct is disclosed herein. In some embodiments, " array " means the scientific tool comprising the association of multiple elements, wherein multiple elements are spatially arranged to allow multiple tests to be carried out on a sample, one or more tests to be carried out on multiple samples, or both. In some embodiments, the array is suitable for screening methods and devices, or is compatible with screening methods and devices (including those relevant to medium or high throughput screening). In further embodiments, the array allows multiple tests to be carried out simultaneously. In further embodiments, the array allows multiple samples to be tested simultaneously. In some embodiments, the array is the microarray of cells. In further embodiments, the microarray of cells is a laboratory tool for allowing multiplex interrogation (multiplex interrogation) of living cells on solid support surfaces. In other embodiments, the array is a tissue microarray. In further embodiments, the tissue microarray includes multiple separated tissues or tissue samples assembled into an array to allow multiple biochemical, metabolic, molecular or histological analysis.

In some embodiments, the engineered liver tissue/construct is each present in a hole of a biocompatible porous container (e.g., see Figure 16). In some embodiments, each tissue is placed in a hole. In other embodiments, each tissue is bioprinted into a hole. In further embodiments, the hole is coated. In various further embodiments, the hole is coated with one or more of the following substances: a biocompatible hydrogel, one or more proteins, one or more chemicals, one or more peptides, one or more antibodies, and one or more growth factors, including combinations thereof. In some embodiments, the hole is coated with NovoGel ^™ . In other embodiments, the hole is coated with agarose. In some embodiments, each tissue is present on a porous biocompatible membrane within a hole of a biocompatible porous container. In some embodiments, each hole of a porous container contains two or more tissues.

In some embodiments, engineered liver tissue/construct is fixed on one or more sides of a biocompatible surface. Many methods are suitable for tissue to be fixed to a biocompatible surface. In each embodiment, tissue is suitably fixed on a biocompatible surface, for example, along one or more overall sides, only at the edge of one or more sides or only at the center of one or more sides. In each further embodiment, tissue is suitably fixed on a biocompatible surface with (being integrated into the surface or being associated with the surface) support or carrier. In each further embodiment, tissue is suitably fixed on a biocompatible surface with (being integrated into the surface or being associated with the surface) one or more clamping clips or plastic patches. In some embodiments, by the attachment of cells to a porous membrane, tissue is suitably fixed on a biocompatible surface. In some embodiments, by being fixed on one or more sides of a biocompatible surface, engineered liver tissue/construct is maintained in an array structure. In a further embodiment, this tissue is attached to a biocompatible surface on 1, 2, 3, 4 or more sides. In some embodiments, the biocompatible surface is any surface that does not pose a significant risk of injury or toxicity to the tissue or organisms in contact with the tissue. In further embodiments, the biocompatible surface is any surface suitable for conventional tissue culture methods. By way of non-limiting example, suitable biocompatible surfaces include treated plastics, membranes, porous membranes, coated membranes, coated plastics, metals, coated metals, glass, treated glass, and coated glass, wherein suitable coatings include hydrogels, ECM components, chemicals, proteins, and the like, and wherein the coating or treatment provides a means of stimulating or preventing cell and tissue adhesion to the biocompatible surface.

In some embodiments, the engineered tissue is fixed to one or more sides of a biocompatible surface to facilitate subjecting the tissue to shear forces caused by fluid flow. In further embodiments, the engineered liver tissue/construct is subjected to shear forces caused by fluid flow. In various embodiments, the engineered liver tissue is subjected to shear forces on 1, 2, 3, 4 or more sides. In further embodiments, the engineered liver tissue/construct undergoes recirculation, perfusion or agitation of liquid nutrients in contact with the tissue on one or more exposed surfaces.

In some embodiments, the array of engineered tissue (including liver tissue/construct) includes the combination of two or more elements. In each embodiment, the array includes 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,125,150,175,200,225,250,275,300,325,350,375,400,425,450,475 or 500 kinds of elements (including any amount therebetween). In further embodiments, each element includes one or more cells, multicellular aggregates, tissue, organ or their combination.

In some embodiments, the array of engineered tissues (including liver tissue/constructs) includes multiple elements arranged in a predetermined pattern space. In further embodiments, the pattern is any suitable spatial arrangement of elements. In various embodiments, as non-limiting examples, the pattern of arrangement includes a two-dimensional grid, a three-dimensional grid, one or more lines, arcs or circles, a series of rows or columns, etc. In further embodiments, the pattern is selected to have compatibility with medium or high throughput biological analysis or screening methods or devices.

In various embodiments, based on specific research purposes or objectives, the cell type and/or source of the cells for manufacturing one or more tissues in the array are selected. In further various embodiments, based on specific research purposes or objectives, the specific tissues in the array are selected. In some embodiments, one or more specific engineered liver tissues are included in the array to promote the research of specific diseases or conditions. In some embodiments, one or more specific engineered liver tissues are included in the array to promote the research of the disease or condition of a specific subject. In further embodiments, one or more specific engineered liver tissues in the array are produced using one or more cell types derived from two or more different human donors. In some embodiments, each tissue in the array is substantially similar in terms of cell type, cell source, cell layer, cell ratio, construction method, size, shape, etc. In other embodiments, one or more tissues in the array are unique in terms of cell type, cell source, cell layer, cell ratio, construction method, size, shape, etc. In various embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300 or more (including any amount therebetween) of the tissues in the array are unique. In other various embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (including any amount therebetween) of the tissues in the array are unique.

In some embodiments, each tissue in the array is independently cultured and maintained. In a further embodiment, the culture conditions of each tissue in the array are such that they are separated from other tissues, and can not exchange culture medium or be dissolved in the factor in the culture medium. In other embodiments, two or more individual tissues in the array exchange soluble factors. In a further embodiment, the culture conditions of two or more individual tissues in the array are such that they exchange culture medium with other tissues or be dissolved in the factor in the culture medium. In each embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300 or more (including any amount therebetween) tissues in the array, exchange culture medium and/or soluble factors. In other various embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (including any amounts in between) of the tissues in the array are exchanged for culture medium and/or soluble factors.

In vitro analysis

In some embodiments, the engineered liver tissues and arrays disclosed herein are used for in vitro analysis. In some embodiments, "analysis" refers to the process of testing or measuring the presence or activity of a substance (e.g., a chemical, molecule, biochemical, drug, etc.) in an organic or biological sample (e.g., a cell aggregate, tissue, organ, organism, etc.). In further embodiments, analysis includes qualitative analysis and quantitative analysis. In further embodiments, quantitative analysis measures the amount of a substance in a sample.

In various embodiments, as non-limiting examples, the engineered liver tissue and arrays are used for image-based analysis, measurement of secreted proteins, expression of markers, and protein production. In various further embodiments, the engineered liver tissue and arrays are used for analysis to detect or measure one or more of: molecule binding (including radioligand binding), molecule absorption, activity (e.g., enzyme activity and receptor activity, etc.), gene expression, protein expression, receptor agonism, receptor antagonism, cell signaling, apoptosis, chemosensitivity, transfection, cell migration, chemotaxis, cell viability, cell proliferation, safety, effectiveness, metabolism, toxicity, infectivity, and abuse liability.

In some embodiments, engineered liver tissue and array are used in immunoassays. In further embodiments, the immunoassay is a competitive immunoassay or a non-competitive immunoassay. In a competitive immunoassay, for example, the antigen in the sample competes with a labeled antigen that is bound to an antibody, and the quantity of the labeled antigen bound to the antibody site is subsequently measured. In a non-competitive immunoassay (also referred to as a "sandwich assay"), for example, the antigen in the sample is bound to the antibody site; then, a labeled antibody is bound to the antigen, and the quantity of the labeled antibody at the site is subsequently measured.

In some embodiments, engineered liver tissue and arrays are used in enzyme-linked immunosorbent assay (ELISA). In further embodiments, ELISA is a biochemical technique for detecting the presence of antibodies or antigens in a sample. In ELISA, for example, at least one antibody specific for a particular antigen is used. For further example, a sample with an unknown amount of antigen is fixed to a solid support (e.g., a polystyrene microplate) non-specifically (by adsorption to a surface) or specifically (in a "sandwich" ELISA, by capturing another antibody specific for the same antigen). For further example, after the antigen is fixed, a detection antibody is added to form a complex with the antigen. The detection antibody is, for example, covalently linked to an enzyme, or self-detected by a second antibody linked to the enzyme via a bioconjugate.

For example, in some embodiments, the array, microarray or chip of cell, multicellular aggregate or tissue is used for drug screening or drug discovery. In further embodiments, the array, microarray or chip of tissue is used as a part of a test kit for drug screening or drug discovery. In some embodiments, each engineered liver tissue/construct is present in the hole of a biocompatible porous container, wherein the container is suitable for one or more automatic drug screening programs and/or devices. In further embodiments, the automatic drug screening program and/or device include any suitable step or device assisted by a computer or automatic machinery.

In further embodiments, arrays for drug screening assays or drug discovery assays are used to research or develop drugs for potential use in any therapeutic area. In still further embodiments, suitable therapeutic areas include, by way of non-limiting example, infectious diseases, hematological diseases, oncology, pediatrics, cardiac diseases, central nervous system diseases, neurology, gastroenterology, liver diseases, urology, infertility, ophthalmology, renal, orthopedic, pain management, psychiatric, pulmonary, vaccines, wound healing, physiology, pharmacology, dermatology, gene therapy, toxicity, and immunity.

In some embodiments, engineered liver tissue and array are used for cell-based screening. In further embodiments, this cell-based screening is for one or more infectious diseases such as viral infection or parasitic infection (for example, Plasmodium infection etc.). In further embodiments, this cell-based screening is for liver fibrosis (for example, sclerosis). In further embodiments, this cell-based screening is for liver cancer. In further embodiments, this cell-based screening is for hepatic steatosis (for example, fatty liver). In further embodiments, this cell-based screening is for one or more metabolic defects. In further embodiments, this cell-based screening is for one or more protein deficiencies. In other embodiments, engineered liver tissue and array are used to study cancer initiation, progression or metastasis. In further embodiments, engineered liver tissue and array are used to study the interaction of other cell types such as cancer cells, cells carrying pathogens, pathogenic cells, immune cells, cells derived from blood or stem cells/progenitor cells and liver tissue and cells comprising liver tissue.

In some embodiments, the construct or its array is used to evaluate the performance of biological products including antibodies, mammalian cells, bacteria, biologically active proteins, hormones, etc. In some embodiments, the construct or its array is used to detect, quantify and study immunological sampling of Kupffer cells and astrocytes, including the effect of signal transduction stimulated by Gram-negative or Gram-positive antigens from Kupffer cells or astrocytes on adjacent hepatocytes (e.g., response to lipopolysaccharide (LPS)). In some embodiments, the construct or its array is useful for the production of hepatotropic viruses including HBV and HCV. In a further embodiment, the construct or its array is used as a vector for producing exoerythrocytic forms of Plasmodium spp. (parasites). In a further embodiment, Plasmodium from liver constructs is used for comparative in vitro analysis to determine effective therapies against exoerythrocytic forms of Plasmodium. In other embodiments, the construct or its array is used as an anti-host therapy for hepatitis C virus (HCV) or Plasmodium, including conducting anti-host antibody studies. In other embodiments, the hepatic construct or array thereof is useful in the study of cancer initiation, progression, or metastasis. In other embodiments, the hepatic construct or array thereof is useful in the study of cell-cell and cell-tissue interactions between mammalian liver cells/tissue, wherein the tissue comprises the construct and one or more additional cell types, including but not limited to pathogen-bearing cells, living pathogenic cells, cancer cells, immune cells, blood cells, stem/progenitor cells, or genetically manipulated cells.

In some embodiments, the array comprises an engineered liver tissue construct and another tissue construct. In a further embodiment, the liver tissue construct is in direct contact with the other tissue construct on one or more surfaces. In a further embodiment, the liver tissue is connected to one or more other tissue constructs or cells via a fluid path or a common reservoir. In a further embodiment, the liquid medium in contact with the engineered liver tissue construct comprises living mammalian cells such as immune cells, cells derived from blood, or cells derived from tumors. In other embodiments, the liquid medium in contact with the engineered liver tissue construct comprises bacteria, viruses, parasites, or other pathogens. In some embodiments, the engineered liver tissue and array are used as carriers to propagate hepatotropic viruses for three-dimensional structural studies including X-ray or cryo-EM.

In vitro support

In some embodiments, the engineered liver tissue construct comprises a plurality of layers, each layer comprising cylindrical bio-inks, the bio-inks being substantially parallel and axially aligned, the bio-inks comprising parenchymal hepatocytes; and optionally, non-parenchymal hepatocytes between the cylindrical bio-inks; and optionally, void spaces or perfusable channels between the cylindrical bio-inks. In further embodiments, the engineered liver tissue is used to provide in vitro support for a subject in need. In still further embodiments, blood is passed through or through the construct to filter the blood and supplement liver function in the subject.

Referring to Figure 17 , in certain embodiments, a bioprinted liver construct is designed to provide in vitro support. In this embodiment, the bioprinted liver construct is composed of multiple cylinders of parenchymal cells. The construct also contains non-parenchymal cells and filler bodies to create compartments and/or channels through which perfusion or flow can be achieved.

19 , in a particular embodiment, a bioprinted liver construct is used as an in vitro liver device comprising a repeating tubular structure having evenly spaced void spaces (depicted as occupied by temporary filler bodies) allowing perfusion across and/or through the engineered liver tissue.

method

In some embodiments, disclosed herein are methods for constructing a living three-dimensional liver tissue construct, the methods comprising bioprinting a bioink comprising at least one liver cell type into or onto a form, and fusing the bioink into a living three-dimensional liver tissue construct. In further embodiments, the tissue construct is for in vitro use.

In some embodiments, methods for constructing tissues (including engineered liver tissues) are also disclosed herein, comprising the steps of: preparing cohesive multicellular aggregates comprising parenchymal or parenchymal and non-parenchymal cells; placing the cohesive multicellular aggregates on a support; and incubating the multicellular aggregates to allow the multicellular aggregates to cohere and form engineered liver tissue; wherein the incubation lasts from about 1 hour to about 30 days. In some embodiments, the method uses bioprinting. In further embodiments, the method, when used, produces engineered liver tissue that is free of or substantially free of preformed scaffolds.

In some embodiments, also disclosed herein is a method for constructing living three-dimensional liver tissue, the method comprising the steps of: preparing one or more cohesive multicellular aggregates comprising mammalian liver cells; placing the one or more cohesive multicellular aggregates on a support; applying one or more of the following to the one or more cohesive multicellular aggregates: a first mammalian cell layer on one or more outer surfaces; a second mammalian cell layer on one or more outer surfaces; and incubating the one or more multicellular aggregates to allow the multicellular aggregates to cohere and form tissue; wherein the incubation lasts for about 1 hour to about 30 days. In some embodiments, the method uses bioprinting. In further embodiments, the method produces engineered liver tissue that is free of or substantially free of a preformed scaffold at the time of use.

In some embodiments, also disclosed herein is a method for constructing a living three-dimensional liver tissue construct, comprising the steps of preparing one or more cohesive multicellular aggregates comprising mammalian cells; placing the one or more cohesive multicellular aggregates on a support to form at least one of: at least one layer comprising a plurality of cell types spatially arranged relative to each other to create a planar geometry, and/or a plurality of layers, at least one layer being compositionally or structurally different from at least one other layer to create a laminar geometry; and incubating the one or more multicellular aggregates to allow the multicellular aggregates to cohere and form a living three-dimensional liver tissue construct.

Preparation of cohesive multicellular aggregates

In some embodiments, the method comprises preparing cohesive multicellular aggregates comprising one or more types of mammalian cells, wherein at least one cellular component represents a hepatocyte. In some embodiments, the method comprises preparing cohesive multicellular aggregates comprising hepatocytes. In some embodiments, the method comprises preparing cohesive multicellular aggregates further comprising non-parenchymal cells. In some embodiments, the method comprises preparing cohesive multicellular aggregates further comprising one or more cell types selected from the group consisting of endothelial cells, astrocytes, fibroblasts, Kupffer cells, immune cells, lymphocytes, cancer cells, virus-carrying cells, pathogenic cells, adipocytes, adipogenic cells, smooth muscle cells, or cells derived from stem/progenitor cells.

There are a variety of ways to prepare multicellular aggregates with the properties described herein. In some embodiments, multicellular aggregates are made from a cell paste containing a plurality of living cells or having a desired cell density and viscosity. In further embodiments, the cell paste is shaped into a desired shape and a multicellular body formed by maturation (e.g., incubation). In some embodiments, the shape of the multicellular body is determined by the shape of the surrounding mold or frame. In further embodiments, the mold or frame is manufactured in a defined geometric shape by an automated instrument. In a further embodiment, the mold or frame is made of bio-ink and contains cells derived from the liver. In a further embodiment, the mold or frame contains non-parenchymal cells derived from the liver and a slurry for filling the frame, and creates a multicellular aggregate containing parenchymal cells. In some embodiments, the multicellular aggregate is substantially cylindrical. In other embodiments, the multicellular aggregate is hexagonal, square, cubic, rectangular, or polyhedral. In a further embodiment, a plurality of multicellular aggregates are formed by making a surrounding mold or frame with a repeating geometric shape in a mosaic pattern and subsequently or simultaneously filling the mold or frame with a cell paste. In a further embodiment, the cell slurry is incubated in a controlled environment to allow the cells to adhere to each other and/or cohere to form elongated multicellular bodies. In another specific embodiment, the multicellular bodies are prepared by shaping a cell slurry comprising a plurality of living cells in a device that holds the cells in a three-dimensional shape. In a further embodiment, the cell slurry is incubated in a controlled environment while being held in a three-dimensional shape for a sufficient time to produce multicellular bodies on a flat surface with sufficient cohesion to support themselves.

In various embodiments, a cell slurry is provided by the following steps: 1) collecting cells (of one or more cell types) or cell aggregates and a biocompatible gel or liquid, such as a cell culture medium (e.g., in a predetermined ratio), to produce a cell suspension; and 2) densifying the cell suspension to produce a cell slurry having the desired cell density and viscosity. In various embodiments, densification is achieved by a variety of methods, such as by concentrating a specific cell suspension obtained by cell culture to obtain the desired cell concentration (density), viscosity, and consistency required for the cell slurry. In specific embodiments, a relatively dilute cell suspension from cell culture is centrifuged for a set time to obtain a concentration of cells in the precipitate that can be formed in a mold. Tangential flow filtration ("TFF") is another suitable method for cell concentration or densification. In some embodiments, a compound is combined with the cell suspension to provide the desired extrusion properties. As non-limiting examples, suitable compounds include: surfactant polyols, collagen, hydrogels, peptide hydrogels, amino acid-based gels, Matrigel ^™ , nanofibers, self-assembling nanofibers, gelatin, fibrinogen, etc.

In some embodiments, a cell slurry is produced by mixing a plurality of living cells with tissue culture medium and densifying the living cells (e.g., by centrifugation). Optionally, one or more ECM components (or derivatives of ECM components) are included by resuspending the cell pellet in one or more physiologically acceptable buffers containing ECM components (or derivatives of ECM components) and re-centrifuging the resulting cell suspension to form a cell slurry.

In some embodiments, the cell density of the cell slurry required for further processing varies with the cell type. In further embodiments, the interactions between cells determine the properties of the cell slurry, and different cell types will have different relationships between cell density and cell-cell interactions. In further embodiments, the cells are pretreated before the cell slurry is formed to increase cell interactions. For example, in some cases, in order to enhance cell-cell interactions before the cell slurry is formed, the cells are cultured in a centrifuge tube after centrifugation. In some embodiments, the cell slurry is formed along with bioprinting; wherein during or after bioprinting, individual cells agglomerate with each other to form bio-ink.

In various embodiments, a variety of methods are used to shape the cell paste. For example, in specific embodiments, the cell paste is manually molded or pressed (for example, after concentration/densification) to obtain the desired shape. For example, the cell paste is absorbed (for example, aspirated) into an instrument such as a micropipette or a syringe (for example, a capillary pipette or a glass/plastic syringe), which shapes the cell paste to conform to the inner surface of the instrument. The cross-sectional shape of the micropipette (for example, a capillary pipette) is alternatively circular, square, rectangular, triangular or other non-circular cross-sectional shapes. In some embodiments, by deposition, the cell paste is shaped in a preformed mold such as a plastic mold, a metal mold or a gel mold. In some embodiments, centrifugal casting or continuous casting is used to shape the cell paste. In some embodiments, the shaping of the bio-ink occurs along with bioprinting, or occurs after bioprinting. In further embodiments, bio-ink formation occurs as a result of co-printing a mold; wherein the mold is optionally deposited by bioprinting; wherein the mold comprises one or more of the following: a bio-ink, a bio-ink further comprising an extrusion compound, a gel, a hydrogel, a synthetic polymer, a sugar, a protein, or mammalian cells, or a combination thereof. In still further embodiments, one or more portions of the co-printed mold are removed after bioprinting; wherein the removal method is selected from the group consisting of: physical means, solubilization with an aqueous medium; chemical treatment; enzymatic treatment; and temperature adjustment. In further embodiments, the co-printed mold remains associated with the tissue after fabrication. In still further embodiments, only the cellular components of the co-printed mold remain associated with the tissue after fabrication.

In some embodiments, multicellular aggregates of defined shapes are also suitable for constructing engineered liver tissues described herein. Spherical multicellular aggregates are optionally formed by a variety of methods, including but not limited to: cell self-assembly, use of molds, and hanging drop methods. In a further embodiment, a method for producing substantially spherical multicellular aggregates comprises the following steps: 1) providing a cell slurry containing a plurality of pre-selected cells or cell aggregates having a desired cell density and viscosity; 2) processing the cell slurry into a cylindrical shape; 3) cutting the cylinder into equal fragments; 4) optionally placing the fragments on a rotary shaker overnight; and 5) optionally maturing the substantially spherical multicellular aggregates over a period of 1 hour to 7 days. In a further embodiment, the multicellular aggregates are formed by an acoustic focusing process.

In some embodiments, partially adhered and/or condensed cell slurry is used for bioprinting; wherein condensation and bio-ink formation mainly occur after printing. In other embodiments, in the first step before bioprinting, the cell slurry is shaped. In a further embodiment, the cell slurry is transferred from a first forming device (e.g., a capillary pipette) to a second forming device (e.g., a mold) that allows the supply of nutrients and/or oxygen to the cells, while they maintain an additional maturation time in the second forming device. An example of a suitable forming device that allows the supply of nutrients and oxygen to the cells is a mold for making multiple multicellular aggregates (e.g., substantially identical multicellular aggregates). For further example, such a mold includes a biocompatible matrix made of a material that resists the migration or ingrowth of cells into the matrix and resists the adhesion of cells to the matrix. In various embodiments, the matrix is suitably made of (PTFE), stainless steel, NovoGel ^™ , agarose, polyethylene glycol, glass, metal, plastic or gel material (e.g., agarose or other hydrogels) and similar materials. In some embodiments, the mold is also suitably configured to allow for the supply of tissue culture medium to the cell slurry (eg, by dispensing the tissue culture medium onto the top of the mold).

Therefore, in an embodiment using a second forming device, the partially adhered and/or condensed cell slurry is transferred from a first forming device (e.g., a capillary pipette) to a second forming device (e.g., a mold). In a further embodiment, the partially adhered and/or condensed cell slurry is transferred by a first forming device (e.g., a capillary pipette) to a groove of a mold. In a further embodiment, after the maturation period (wherein the mold is incubated in a controlled environment with the cell slurry retained therein so that the cells in the cell slurry further adhere to each other and/or condense to form multicellular aggregates), the cohesion of the cells will be strong enough to allow the resulting multicellular aggregates to be picked up with a tool (e.g., a capillary pipette). In a further embodiment, the capillary pipette is suitably part of a print head of a bioprinter or similar device, which can be operated to automatically place the multicellular aggregates in a three-dimensional construct.

In some embodiments, the cross-sectional shape and size of the multicellular aggregates substantially correspond to the cross-sectional shape and size of the first forming device and the optional second forming device used to prepare the multicellular aggregates, and a person skilled in the art will be able to select an appropriate forming device having an appropriate cross-sectional shape, cross-sectional area, diameter and length that is suitable for producing multicellular aggregates having the cross-sectional shape, cross-sectional area, diameter and length discussed above.

Placing the cohesive multicellular aggregates on a support

Many methods are suitable for placing multicellular aggregates on a support to produce a desired three-dimensional structure. For example, in some embodiments, the multicellular aggregates are manually placed in contact with each other, deposited in place by extrusion from a pipette, nozzle, or needle, or placed by automated computer-assisted means (e.g., a bioprinter).

As described herein, in various embodiments, the multicellular aggregates have many suitable shapes and sizes. In some embodiments, the multicellular aggregates are elongated and have any of several suitable cross-sectional shapes, including, by way of non-limiting example, circular, elliptical, square, triangular, polygonal, and irregular shapes. In further embodiments, the multicellular aggregates are elongated and are in the form of cylinders. In some embodiments, the elongated multicellular aggregates have similar lengths and/or diameters. In other embodiments, the elongated multicellular aggregates have different lengths and/or diameters. In some embodiments, the multicellular aggregates are substantially spherical. In some embodiments, the engineered liver tissue comprises substantially spherical multicellular aggregates that are substantially similar in size. In other embodiments, the engineered liver tissue comprises substantially spherical multicellular aggregates that have different sizes. In some embodiments, engineered liver tissues of different shapes and sizes are formed by arranging multicellular bodies of various shapes and sizes.

In some embodiments, the coagulated multicellular aggregates are placed on a support. In various embodiments, the support is any suitable biocompatible surface. In further embodiments, suitable biocompatible surfaces include, by way of non-limiting example, polymeric materials, porous membranes, plastics, glass, metals, hydrogels, and combinations thereof. In some embodiments, the support is coated with a biocompatible substance, by way of non-limiting example, a hydrogel, a protein, a chemical, a peptide, an antibody, a growth factor, or a combination thereof. In one embodiment, the support is coated with NovoGel ^™ . In another embodiment, the support is coated with agarose. In one embodiment, the coagulated multicellular aggregates are placed in a well of a biocompatible porous container.

In some embodiments, once placement of the cohesive multicellular aggregates is complete, tissue culture medium is poured on top of the construct. In further embodiments, tissue culture medium enters the spaces between the multicellular bodies to support the cells in the multicellular bodies.

Applying a layer of a first cell type and/or a layer of a second cell type

A variety of methods are suitable for applying one or more cell layers to one or more external surfaces of the coagulated mammalian cell structure. For example, in some embodiments, applying the cell layer comprises coating one or more surfaces of the coagulated multicellular assembly with a suspension, sheet, monolayer, or fused aggregate of cells. In various embodiments, one, two, three, four, or more surfaces of the coagulated mammalian cell construct are coated.

In some embodiments, applying the cell layer comprises: bioprinting an additional layer of fused multicellular aggregates. In other embodiments, applying the cell layer comprises: bioprinting, spraying, or ink-jetting a solution, suspension, or liquid concentrate of cells. In further embodiments, a suitable cell suspension comprises about 1 x 10 ⁴ to about 1 x 10 ⁶ cells/μl. In still further embodiments, a suitable cell suspension comprises about 1 x 10 ⁵ to about 1.5 x 10 ⁵ cells/μl. In further embodiments, applying the cell layer comprises: dispensing the suspension of cells in the form of spatially distributed droplets directly onto one or more surfaces of the coagulated mammalian cell structure. In still further embodiments, applying the cell layer comprises: dispensing the suspension of cells in the form of a spray directly onto one or more surfaces of the coagulated mammalian cell structure. In various embodiments, the cell layer is applied at any suitable time during the construction process. In some embodiments, one or more cell layers are applied to one or more outer surfaces of the coagulated mammalian cell construct immediately after bioprinting (e.g., up to 10 minutes). In other embodiments, one or more layers are applied after bioprinting (e.g., after 10 minutes). In yet other embodiments, one or more layers are applied during maturation of the structure.

In some embodiments, the method further includes the step of culturing a cell layer on a support. In such embodiments, in some cases, applying the cell layer includes placing one or more surfaces of the engineered liver tissue construct in direct contact with the prepared cell culture medium. In further embodiments, the construct is bioprinted directly on a culture layer of cells or on a cell monolayer. Any type of cultured cell layer on a biocompatible support is suitable. In some embodiments, multicellular aggregates are bioprinted onto an endothelial cell layer. In other embodiments, multicellular aggregates are bioprinted onto a non-parenchymal cell layer. In further embodiments, the cell layer adheres and/or agglomerates to the multicellular aggregates of the bioprinted construct. In some embodiments, each layer of the multilayer structure is bioprinted. In further embodiments, individual layers include bio-inks in variable forms, including but not limited to: agglomerated cell aggregates, cell slurries, cell slurries combined with extrusion compounds or other additives, cell monolayers, and cell sheets.

Co-printed molds for fabricating compartmentalized tissues

In some embodiments, the method includes preparing one or more bio-inks comprising non-parenchymal cells; preparing one or more bio-inks comprising parenchymal cells, such as hepatocytes or hepatocyte-like cells; depositing the bio-inks onto a support; and incubating the deposited bio-inks for about 1 hour to about 30 days to form a living three-dimensional liver tissue construct comprising at least one compartment comprising an interior comprising parenchymal cells bounded by a side comprising non-parenchymal cells.

In some embodiments, the method includes preparing one or more cohesive multicellular aggregates comprising mammalian hepatocytes; placing the one or more cohesive multicellular aggregates on a support to form at least one of: at least one layer comprising a plurality of cell types spatially arranged relative to each other to create a planar geometry; and a plurality of layers, at least one layer being compositionally or structurally different from at least one other layer to create a laminar geometry; and incubating the one or more multicellular aggregates for a period of about 1 hour to about 30 days to allow them to cohere and form a living three-dimensional liver tissue construct.

Referring to Figure 2, in a specific embodiment, a liver construct containing HepaRG cells was bioprinted, matured, and biochemically characterized. First, a perimeter box with dimensions of 4 mm x 4 mm x 1 mm was printed using a bioink containing endothelial cells and hepatic stellate cells. Second, HepaRG cells were deposited in the center of the perimeter box. The cells were then matured at 37°C for 96 hours. Third, the bioprinted construct was observed for fusion 20 hours after printing. In this embodiment, the bioprinted liver construct was metabolically active, producing albumin and cholesterol at 96 hours.

3 , in a particular embodiment, a liver construct was bioprinted using a non-parenchymal cell rim and a parenchymal cell filler, each made from an extruded compound.

4 , in a particular embodiment, a liver construct is bioprinted using a rim comprising endothelial cells and hepatic stellate cells, a filler comprising primary hepatocytes, and a third component introduced as spatially dispersed spheroids comprising endothelial cells and hepatic stellate cells within the filler.

5 , in certain embodiments, a liver construct is bioprinted using a co-mold made of a cell-free material that dissolves in an aqueous medium (eg, PF-127), leaving only the filler and creating negative space.

6 , in certain embodiments, liver constructs were bioprinted using a continuous deposition technique using a bio-ink composed of multiple liver cell types encapsulated in a water-soluble extrusion compound (e.g., PF-127) to form a mosaic of functional unit patterns.

8, in a particular embodiment, a compartmentalized liver construct was bioprinted using a co-printed mold that did not contain cells used to shape the multicellular aggregates into hexagons. In this embodiment, the acellular material disappears upon use to produce a scaffold-free tissue.

10 , in a particular embodiment, a compartmentalized liver construct was bioprinted using a co-mold that served as the sides populated with HepG2 cells.

11 , in a particular embodiment, the co-printed mold containing cells is bioprinted as a side portion in which parenchymal cells serve as the filler, in this case, HepG2 cells.

12 , in certain embodiments, compartmentalized liver constructs were bioprinted using a non-parenchymal cell border and a parenchymal cell fill to achieve high, tissue-like density.

Incubation of multicellular aggregates

In some embodiments, multicellular aggregates are incubated. In further embodiments, incubation allows multicellular aggregates to adhere and/or condense to form tissue, such as liver tissue. In some embodiments, in a cell culture environment (e.g., culture dish, cell culture flask, bioreactor, etc.), multicellular aggregates condense to form tissue. In further embodiments, in an environment with conditions suitable for promoting the growth of cell types included in the multicellular aggregates, multicellular aggregates condense to form tissue. In one embodiment, at about 37 ° C, in a humidified atmosphere containing about 5% CO ₂ , in the presence of a cell culture medium containing factors and/or ions that promote adhesion and/or condensation, multicellular aggregates are incubated. In other embodiments, the multicellular aggregates are maintained in an environment containing 0.1% to 21% O ₂ .

In various embodiments, incubation has any suitable duration. In further various embodiments, incubation has a duration of about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180 or more minutes, including any amount therebetween. In further various embodiments, incubation has a duration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48 or more hours, including any amount therebetween. In further various embodiments, the incubation has a duration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days, including any amount therebetween. Several factors influence the time required for multicellular aggregates to cohere to form tissue, including, by way of non-limiting example, cell type, ratio of cell types, culture conditions, and the presence of additives such as growth factors.

Additional steps to increase viability of engineered tissues

In some embodiments, the method further comprises the step of increasing the viability of the engineered tissue. In further embodiments, these steps comprise providing direct contact between the tissue and the nutrient medium via a temporary or semi-permanent grid of confinement material. In some embodiments, the tissue is confined within a porous or interstitial material. Direct access of at least some cells of the engineered tissue to nutrients increases the viability of the engineered tissue.

In further embodiments, additional and optional steps for increasing the viability of engineered tissues include: 1) optionally dispensing a base layer of confinement material prior to placing the cohesive multicellular aggregates; 2) optionally dispensing a perimeter of confinement material; 3) bioprinting cells of the tissue within a defined geometry; and 4) dispensing elongated bodies (e.g., cylinders, ribbons, etc.) of confinement material covering the primary tissue in a pattern that introduces gaps in the confinement material, such as a grid, mesh, or lattice.

Many restriction materials are suitable for use in the methods described herein. In some embodiments, hydrogel is an exemplary restriction material having one or more advantageous properties, including: non-adherent, biocompatible, extrudable, bioprintable, non-cellular, with suitable strength and insoluble in aqueous conditions. In some embodiments, suitable hydrogel is a natural polymer. In one embodiment, the restriction material is composed of NovoGel ^™ . In a further embodiment, suitable hydrogels include hydrogels such as Pluronic F-127, collagen, hyaluronate, fibrin, alginate, agarose, chitosan, gelatin, and derivatives or combinations thereof derived from surfactant polyols. In other embodiments, suitable hydrogels are synthetic polymers. In a further embodiment, suitable hydrogels include those derived from poly (acrylic acid) or derivatives thereof, poly (ethylene oxide) and copolymers thereof, poly (vinyl alcohol), polyphosphazene, and combinations thereof. In various specific embodiments, the confinement material is selected from the group consisting of: hydrogel, NovoGel ^™ , agarose, alginate, gelatin, Matrigel ^™ , hyaluronic acid, poloxamer, peptide hydrogel, poly(isopropyl n-polyacrylamide), polyethylene glycol diacrylate (PEG-DA), hydroxyethyl methacrylate, polydimethylsiloxane, polyacrylamide, poly(lactic acid), silicon, silk, and combinations thereof.

In some embodiments, the gaps in the overlay pattern are evenly or substantially evenly distributed around the surface of the tissue. In other embodiments, the gaps are unevenly distributed, thereby unevenly exposing the cells of the tissue to nutrients. In uneven embodiments, differential access to nutrients is optionally exploited to influence one or more properties of the tissue. For example, in some cases, it may be desirable for cells on one surface of a bioprinted tissue to proliferate faster than cells on another surface of the bioprinted tissue. In some embodiments, the exposure of various portions of the tissue to nutrients is optionally varied at different times to influence the development of the tissue toward a desired endpoint.

In some embodiments, the confinement material is removed at any suitable time, including but not limited to: immediately after bioprinting (e.g., within 10 minutes), after bioprinting (e.g., after 10 minutes), before the cells are substantially coagulated with each other, after the cells are substantially coagulated with each other, before the cells produce extracellular matrix, after the cells produce extracellular matrix, just before use, etc. In various embodiments, the confinement material is removed by any suitable method. For example, in some embodiments, the confinement material is cut, torn off from the cells, digested, or dissolved.

In some embodiments, the method further comprises the step of subjecting the engineered liver tissue to shear forces on one or more sides caused by fluid flow, agitation, or convective recirculation.

Biological properties

The bioprinted liver tissue described herein comprises a multicellular structure similar to human tissue. Like native liver tissue, the bioprinted liver tissue contains specific cell types (e.g., hepatic stellate cells, hepatocytes, endothelial cells, etc.) enriched in specific areas, resulting in a patterned, compartmentalized structure.

In some embodiments, the bioprinted liver tissue described herein comprises important features of a native liver, including high cell density, and production of ECM, lipids, and glycogen. See Figure 21. In some embodiments, the bioprinted liver tissue described herein has a cell density of at least 30%. In various further embodiments, the bioprinted liver tissue described herein has a cell density of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In various further embodiments, the bioprinted liver tissue described herein has a cell density of about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%.

In some embodiments, the three-dimensional bioprinted liver tissue comprises histological features such as tight junctions, maintenance of at least some quiescent hepatic stellate cells, and development of microvascular structures. In some embodiments, the three-dimensional bioprinted liver tissue comprises compartmentalization that persists well after initial fabrication. For example, in further embodiments, the three-dimensional bioprinted liver tissue comprises compartmentalization that persists for at least 7 days, at least 10 days, at least 20 days, at least 30 days, or at least 40 days. In various further embodiments, the three-dimensional bioprinted liver tissue comprises compartmentalization that persists for about 7 days, about 10 days, about 20 days, about 30 days, or about 40 days.

In some embodiments, after fabrication, regions of hepatocytes surrounded by non-parenchymal "stroma" can be identified. See Figure 22. Unexpectedly, the bioprinted liver tissue showed significant self-organization into lobular structures that closely mimicked native liver. The observed structures supported function and viability for over 40 days, enabling long-term testing, e.g., low doses, repeated exposures, and chronic pathological conditions.

In some embodiments, liver-specific functions are maintained for more than 7, 10, 20, 30, or 40 days in the three-dimensional bioprinted liver tissues described herein. For example, in some embodiments, the bioprinted liver tissues described herein show continuous production of albumin for up to 6 weeks (42 days), far exceeding the time course of primary hepatocytes published in traditional two-dimensional techniques. See Figure 23. The bioprinted liver tissues also show continued production of other hepatocyte factors such as cholesterol. See Figure 23.

3D bioprinted human liver tissue demonstrated production of other relevant metabolites, including transferrin and fibrinogen, that were not achieved using existing 2D methods. See Figure 24. In some embodiments, 3D bioprinted human liver also demonstrated markers of bile duct formation. See Figure 25.

In some embodiments, the three-dimensional bioprinted liver tissue constructs described herein exhibit sustained inducibility of active CYP3A4 enzymes, compared to two-dimensional primary hepatocytes that lose function after several days, wherein the inducibility is inhibited by verapamil. See Figure 26. In some embodiments, the three-dimensional bioprinted liver tissues described herein exhibit native-like responses to known liver toxicants, including diclofenac, trovafloxacin, and methotrexate. See Figures 27-29. In some embodiments, the three-dimensional bioprinted liver tissues described herein exhibit native-like responses to physiologically relevant molecules such as vitamin D. See Figure 30.

In some embodiments, the three-dimensional human liver tissue described herein is patterned with parenchymal cells (hepatocytes) and non-parenchymal cell regions (endothelial cells (EC) and hepatic stellate cells (hSC)). In further embodiments, as an alternative to primary hepatocytes, the patterned three-dimensional liver tissue includes iPSC-derived hepatocyte-like cells (iPSC-HC) (e.g., iCell Hepatocytes, Cellular Dynamics Inc.). The use of iPSCs allows the development of patient-specific and/or disease-specific in vitro systems and can provide a means of generating cells that are difficult to isolate or propagate.

Example

The following specific embodiments should be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way.

Example 1 - Bioprinting of liver tissue using sequential deposition and mosaic functional unit structures

Engineered liver tissue was bioprinted using a continuous deposition mechanism using a NovoGen MMX Bioprinter ^™ (Organovo, Inc., San Diego, CA). The three-dimensional structure of the liver tissue is based on repeating functional units, in this case hexagons. The bioink consists of hepatic stellate cells and endothelial cells encapsulated in an extrusion compound (surfactant polyol - PF-127).

Preparation of 30% PF-127

A 30% PF-127 solution (w/w) was prepared using PBS. PF-127 powder was mixed with cooled PBS using a magnetic stir plate maintained at 4° C. Complete dissolution occurred in approximately 48 hours.

Cell preparation and bioprinting

A cell suspension consisting of 82% hepatic stellate cells (SC) and 18% human aortic endothelial cells (HAEC) and human adult dermal fibroblasts (HDFa) was divided into 15 mL tubes to obtain three cell concentrations after centrifugation: ^50x106 cells/ml, ^100x106 cells/ml, and ^200x106 cells/ml. Each cell pellet was resuspended in 30% PF-127 and aspirated into a 3cc reservoir used by a bioprinter. Using a 510 μm dispensing tip, the encapsulated cells were bioprinted onto a PDMS substrate as a single hexagon (see Figure 6A) or a hexagonal mosaic structure (see Figure 6B). Each construct received approximately 200 μL of culture medium and was incubated at room temperature for 20 minutes to evaluate the integrity of the construct.

Multilayer bioprinting

For the hexagonal mosaic experiment, bioprinting was performed up to (4) consecutive layers, resulting in a taller structure with more cellular material present. After fabrication, each construct initially received approximately 200 μL of complete medium to assess the integrity of the construct. The construct was incubated at room temperature for 20 minutes and then immersed in 20 mL of complete medium.

result

After 18 hours of culture in growth medium containing 10% fetal bovine serum (which dissolves PF127), the cells contained within the bioprinted geometry were coagulated to produce a complete, contiguous sheet of tissue that maintained the geometric pattern of the original design (see Figure 6D). Figure 7 shows an H&E staining of a single segment of the mosaic construct after fixation in 10% neutral buffered formalin. The cells were found to be viable, intact, and confined to their original printed geometry.

Example 2 - Forced Stratification

Cell populations (homogeneous or heterogeneous) for bioprinting were prepared in Pluronic F-127 (Lutrol, BASF) in the form of cylindrical bio-inks or cell suspensions. Briefly, for bio-ink preparation, cells were released from standard tissue culture plastic using recombinant human trypsin (75 μg/mL, Roche) or 0.05% trypsin (Invitrogen). After enzyme release, the cells were washed, collected, counted, and combined in the desired ratio (i.e., 50:50 hepatic stellate cells (hSC): endothelial cells (EC)) and pelleted by centrifugation. The supernatant was then removed from the cell pellet, and the cell mixture was aspirated into a glass microcapillary tube of the desired diameter (typically 500 μm or 250 μm inner diameter). This cylindrical cell preparation was then extruded into a mold formed from a non-cell-adhesive hydrogel material with pores for bio-ink maturation. The resulting bio-ink cylinders are then cultured in complete cell culture medium for an empirically determined time, typically 2 to 24 hours.

In brief, for the preparation of hydrogel cell suspensions, cells were released from standard cell culture containers using any of the enzyme-mediated protocols described herein. Subsequently, the released cells were washed, collected, counted, and centrifuged with serum-containing culture medium to form a dense cell pellet. The supernatant was removed from the resulting cell pellet and then resuspended in cold PF-127 (4°C) at a concentration of 50 to 200 x ¹⁰⁶ cells/mL (range of 10-300 x ¹⁰⁶ cells/mL). The cell suspension was then drawn into a syringe using a NovoGen MMX Bioprinter ^™ bioprinter (Organovo, Inc., San Diego, CA).

Subsequently, a bioprinter is utilized to complete the production of a tissue construct with forced cell patterning, stratification or orientation. Using cylindrical bio-ink, a cell suspension in a water-soluble gel, or a combination thereof, the bioprinting of a three-dimensional tissue construct is performed. In order to obtain a defined cell pattern or stratification, a combination of related cell populations is included in the bio-ink or cell suspension preparation, and then bioprinting is performed in a manner that dissolves the gel material of the loaded cell solution, thereby producing a defined cell stratification around the deposited bio-ink (see Figure 13). Cell patterning, organization, or stratification can also be obtained by utilizing and incorporating defined, discrete cell populations (e.g., hSC and EC), which results in the formation of predictable and repeatable organization of cells and cell structures within the bioprinted tissue (see Figure 14).

In some experiments, the final cell organization structure in the bioprinted new tissue was observed after a maturation or culture period. The constructs were maintained in a standard laboratory incubator (37°C, humidified chamber supplemented with 5% _CO2 ) and evaluated over time.

result

Bioprinting enables cell patterning, layering, or placement. Different cell tissue structures are observed by bioprinting bioinks containing heterogeneous (i.e., polymorphic) cell populations, or by combining bioinks (homogeneous or heterogeneous cell populations) with high-density cell gels or cell suspensions. These new tissue constructs mature in a humidified chamber (incubator), leading to the further establishment of different cell arrangements, structures, and/or segregations within these bioprinted new tissues.

For example, bioprinting of EC:hSCs loaded with PF-127 on bioprinted bioinks containing HepG2 cells resulted in the creation of distinct layers of constructs with distinct cell populations and discrete tissue morphologies. In the case of bioink constructs containing both hSCs and ECs, bioprinted constructs grown in complete medium for more than 3 days were found to contain distinct EC layers at the periphery and organized microtubule networks in the core of the construct. Bioprinted constructs made with bioinks containing a homogeneous (i.e., monotypic) population of vascular smooth muscle cells, onto which a high concentration EC solution was bioprinted, were found to contain distinct EC layers at the periphery of the construct.

Example 3 - Multi-well plate

Cell populations (homogeneous or heterogeneous) were prepared for bioprinting using various bioink formats, including cylindrical bioink aggregates, suspensions of cell aggregates, or cell suspensions/slurries, optionally containing extrusion compounds. Briefly, to prepare cylindrical bioinks, cells were released from standard tissue culture plastic using recombinant human trypsin (75 μg/mL, Roche) or 0.05% trypsin (Invitrogen). Following enzymatic release, the cells were washed, collected, counted, and combined at the desired ratio (i.e., 50:50 hepatic stellate cells (hSC): endothelial cells (EC)) and pelleted by centrifugation. The supernatant was then removed from the cell pellet, and the cell mixture was aspirated into a glass microcapsule of the desired diameter (typically 500 μm or 250 μm inner diameter). This cylindrical cell preparation was then extruded into a mold formed from a non-cell-adhesive hydrogel material with pores for bioink maturation. The resulting bio-ink cylinders are then cultured in complete cell culture medium for an empirically determined time, typically 2 to 24 hours.

To prepare a cell suspension or cell slurry of cell aggregates, the cell propagation and release protocols described herein were followed. Upon generating a cell pellet, the supernatant was removed from the pellet and transferred to a conventional deposition syringe. This syringe was then mounted on a bioprinter deposition head for direct bioprinting of the cell aggregate solution or slurry into a multiwell plate.

The replicated tissue constructs are then bioprinted into wells of multi-well tissue culture plates (e.g., 6-well or 24-well) or multi-well cell culture inserts (i.e., Transwell, BD) and then placed into appropriate multi-well plates. After bioprinting, the three-dimensional constructs are allowed to mature/condition with the relevant culture medium for a period of time, typically 3 to 14 days. After maturation, the constructs are harvested, fixed, and processed for routine histology and immunohistochemistry.

result

Bioprinted tissues were successfully fabricated in multiwell culture plates or multiwell culture inserts (which were subsequently inserted into multiwell plates). This approach allows for the generation of replicate bioprinted tissues that can be cultured and processed to create identical or unique culture conditions. This approach results in a significant increase in the throughput and sample generation of the bioprinting process (see Figure 16).

Example 4 - Stimulation of bioprinted new tissue

Cylindrical bio-inks comprising relevant heterogeneous (i.e., polymorphic) cell populations were prepared. Physiologically relevant populations of cells (e.g., hepatic stellate cells (hSC) and human aortic endothelial cells (EC)) were combined in specific ratios to produce suitable bio-inks. The cells were maintained and propagated under standard laboratory conditions and cultured in the following culture media: culture media purchased from the same supplier as the cells, or culture media that included components generally found in the original literature that were favorable for standard cell culture practices for those specific cell types. Cells were processed for bio-ink preparation as follows: Briefly, cells were released from standard tissue culture plasticware by washing with cation-free phosphate-buffered saline (PBS), and then exposed to 0.1-0.05% trypsin (Invitrogen). The released cells were then washed with serum-containing culture medium, collected, counted, and (for stimulation analysis or ongoing experiments) combined in appropriate ratios and precipitated by centrifugation. The supernatant was then removed, and the cell pellet was aspirated into a glass microtube, which was then immersed in complete culture medium for approximately 15 to 20 minutes. The cylindrical bio-ink structure was then extruded into a non-cell-adhesive hydrogel mold (containing linear channels) and maintained for 2 to 18 hours. Bioprinted tissue constructs were then made using cylindrical bio-inks containing hSC:ECs and maintained and/or matured in a humidified chamber. Tissue segments with an initial size of 1.25 mm x 8 mm x 0.25 mm (W x L x H) were fabricated. During post-printing maturation, some constructs were exposed to the cytokine TGF-β1 to elicit tissue-specific responses (see Figure 15).

For the tissue construct requiring homogeneous (i.e., single type) cell layer, it is limited to the upper surface and the second cell preparation is carried out using a cell type containing related cells. Typically, in a high concentration cell suspension, vascular endothelial cells or small airway epithelial cells (respectively for vascular wall and human lung tissue model) are prepared. In brief, as described above, cells are released, collected, counted and precipitated by centrifugation. The supernatant is removed and the cell pellet is resuspended in a small volume of complete medium to produce a high concentration cell mass of 1x10 ⁵ cells/μL. Subsequently, the cell suspension is stored at 4°C until use.

The bioprinted neotissues were then immersed in complete cell culture medium containing serum and placed in a standard humidified chamber supplemented with 5% _CO2 to mature. Subsequently, the bioprinted neotissues were cultured and stimulated with relevant cytokines for a predetermined time, formalin-fixed, harvested, and processed for routine histology and immunohistochemistry. The bioprinted tissues were evaluated for characteristics such as, but not limited to, tissue morphology, cellularity, extracellular matrix production, cell proliferation, cell viability, and construct integrity.

Cytokine stimulation was performed by adding cytokines directly to the culture medium and culturing the bioprinted tissue with the added protein to provide cells with direct and prolonged access to appropriate stimuli. Dose-response experiments were generally performed at doses of 0.1 to 100 ng/mL (depending on the ED50 of the experimental cytokine). For experiments in which cytokine stimulation was performed for more than 48 hours, the culture medium was changed and fresh cytokines were added every 48 hours.

result

Bioprinted neotissues containing physiologically relevant cell populations were successfully stimulated with cytokines that had previously been shown to elicit cellular responses in two-dimensional in vitro systems. The responses observed in the bioprinted three-dimensional tissue constructs were found to be dose-dependent and limited to cells within the bioprinted constructs (see, e.g., FIG15 ).

Example 5 - Bioprinting co-formed functional liver tissue microstructures using sequential deposition

Preparation of 30% PF-127

Prepare a 30% PF-127 solution (w/w) in PBS. Mix PF-127 powder with cold PBS using a magnetic stir plate maintained at 4°C. Complete dissolution occurs in approximately 48 hours.

Co-printing and filling of cell preparation and molds

Using a bioprinter and a 510 μm dispensing tip, 3 mL of PF-127 solution was aspirated into a 3 cc reservoir. A 30% PF-127 solution was bioprinted into a 6-well Transwell to form a single hexagon and layered six times.

A cell suspension consisting of ^7.8x107 hepatocytes (HepG2) was centrifuged at 1000g for 6 minutes to produce a cell slurry. 5 μL of cell slurry was squeezed out through a 510 μm needle to fill each triangular mold (see Figure 5A). The hexagonal mold was incubated at room temperature for 15 minutes. 3 mL of culture medium (DMEM supplemented with 10% FBS and 1X penicillin, streptomycin and amphotericin B) and the Transwell loaded as above were added to the hole and then incubated at 37 ° C and 5% _CO2 . Within 45 minutes, the PF-127 mold was dissolved in the culture medium, leaving a complete molded liver bio-ink to form a planar geometry of cells and gaps (see Figure 5B). To remove residual PF-127 from the culture medium, the Transwell was transferred to a new hole containing 3 mL of culture medium and incubated for two hours. This operation was repeated twice more, so that a total of 9 mL of culture medium exchange was performed over 6 hours.

After 6 hours, the Transwell was transferred to a new well without culture medium and a cell suspension of 2x10 cells was distributed at a ratio of 90% human aortic endothelial cells and ¹⁰ % hepatic stellate cells to fill the gaps formed by the dissolution of the FP-127 mold. The liver construct was incubated at room temperature for 15 minutes. After 15 minutes of incubation, 4 mL of culture medium containing 85% culture medium (DMEM supplemented with 10% FBS and 1X penicillin, streptomycin and amphotericin B to load hepatocytes and hepatic stellate cells; and 15% M199 supplemented with 2% LSGS, 10% FBS, HEPES and 1X penicillin, streptomycin and amphotericin B to load human aortic endothelial cells) was added. The construct was incubated at 37°C and 5% _CO2 for 48 hours to form a continuous construct having a planar geometry of a small leaflet (triangular) structure of the liver parenchyma that was inserted into the tissue comprising endothelial cells.

Example 6 - Bioprinting and Characterization of Functional Liver Tissue

Cell culture

Cryopreserved primary human hepatocytes (Life Technologies) were purified according to the manufacturer's protocol and incubated in Hepatocyte Maintenance Media (Life Technologies) supplemented with penicillin 100 I.U./ml, streptomycin 0.25 μg/ml, and amphotericin 85 μg/ml (1XP/S/A) (Life Technologies). The following cells were used in the non-parenchymal cell component of the bioprinted tissue: human umbilical vein endothelial cells (HUVEC; Becton Dickenson) and hepatic stellate cells (ScienCell) and propagated according to the manufacturer's instructions. HUVECs were grown in EGM-2 medium (Lonza), while hepatic stellate cells were grown in DMEM (Corning) supplemented with 10% FBS (Life Technologies), penicillin 100 I.U./ml, streptomycin 0.25 μg/ml, and amphotericin 85 μg/ml (1XP/S/A; Corning).

Chemicals used in induction assays

The following reagents were purchased from Sigma: rifampicin, diclofenac, verapamil, 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate, and vitamin D3.

Bioink preparation

Bioinks were prepared based on the protocols and techniques described herein. To prepare cell-containing hydrogels, NovoGel 2.0 (Organovo, Inc.) was prepared according to the manufacturer's instructions and a non-parenchymal cell population was introduced prior to bioprinting.

Bioprinting

All tissues were directly manufactured onto the membranes of standard tissue culture well inserts (Corning Transwell) using the standard Organovo bioprinting protocol and the NovoGen MMX Bioprinter or its improved version. The bioprinted liver tissue includes a side containing non-parenchymal cells (hepatic stellate cells, HUVEC) in Novogel 2.0. The side defines a compartment containing an interior. The interior of the compartment is filled with a cell slurry containing primary hepatocytes. The bioprinted liver tissue also includes a groove (moat) comprising Novogel 1.0. Figure 20 shows a specific example of bioprinted human liver tissue. During the experiment, the bioprinted liver tissue remained on these membranes. The constructs were cultured daily with a 600 μl mixture of 90% primary hepatocyte culture medium and 10% EGM-2 and incubated at 37°C under humidified air conditions supplemented with 5% CO _2. The exhausted culture medium was collected for analysis of secreted factors, and liver tissue was harvested for endpoint analysis (e.g., gene expression, histology, tissue viability, etc.).

Secreted protein detection

Spent culture medium was collected and stored at -80°C and analyzed for the following hepatic metabolites by commercially available ELISA kits: albumin (Bethyl Laboratories), fibrinogen (GenWay Labs), and transferrin (GenWay Labs). Cholesterol biosynthesis was quantified using a fluorometric method (Cayman Biochemical). Lactate dehydrogenase (LDH) activity was measured colorimetrically using a commercially available reagent (Abcam).

RNA isolation and cDNA synthesis for transcriptional profiling

Use Quick-RNA MiniPrep (Zymo) to extract a large amount of RNA from three-dimensional liver construct according to the manufacturer's instructions. Use spectrophotometer (Beckman Coulter DU 640) to quantify RNA. According to the manufacturer's instructions (CloneTech), 2 μg of input RNA is used in each independent cDNA synthesis SmartScribe reaction. According to the manufacturer's guidance (Life Technologies), the TaqMan (Life Technologies) probe and primer pair for CYP3A4 and VIC dye labeling for 18S of FAM dye labeling are used in TaqMan Universal Master Mix UNG. Use Step One Plus system (Life Technologies) to detect transcription amplicon.

Histological analysis: paraffin preparation of liver tissue

The liver constructs were fixed in situ for 24 hours in a 2% paraformaldehyde solution (2% paraformaldehyde, 10mM calcium chloride, 50mM sucrose in PBS) at room temperature. After 24 hours, the fixative solution was removed and replaced with 70% ethanol. The constructs with Transwell membranes were immersed in 45°C liquid Histogel (American MasterTech, Lodi, CA, catalog number EMHGECS) in a plastic biopsy mold and allowed to solidify at room temperature. The Histogel blocks with embedded liver constructs were then processed for paraffin embedding using a tissue processor. After paraffin infiltration, the liver constructs were embedded in a paraffin mold and a rotary microtome (Jung Biocut 2035, Leica Microsystems, Buffalo Grove, IL) was used to generate 5 μm cross-sections. For hematoxylin and eosin staining, the slides were dewaxed in xylene and rehydrated with 100%, 95%, 70%, and 50% ethanol and rinsed in distilled water. Slides were immersed in Gill's hematoxylin (Fisher Scientific, Pittsburgh, PA). After rinsing with distilled water, the slides were briefly immersed in 0.2% v/v ammonium hydroxide. After rinsing with distilled water, the slides were immersed in an aqueous eosin solution (American MasterTech). The slides were then dehydrated through an ethanol gradient, rinsed in xylene, and mounted with resinous mounting media (CytoSeal, Fisher Scientific).

Periodic acid-Sciff staining was performed according to the manufacturer's instructions (Abcam, Cambridge, MA).

For immunohistochemical analysis, slides were dewaxed in xylene and rehydrated by sequentially immersing them in 100%, 95%, 70%, and 50% ethanol before a final wash in distilled water. The solution and slides were heated to near boiling using a standard microwave oven, followed by slow cooling for 30 minutes, and the rehydrated sections were subjected to heat-mediated antigen retrieval in 10 nM sodium citrate pH 6.0. The slides were then blocked with 10% goat serum in Tris-buffered saline (TBS) for 1 hour and then incubated with the primary antibody overnight at 4°C. The following primary antibodies were used: rabbit anti-E-cadherin (Abcam; 1:50); mouse anti-albumin (Sigma-Aldrich, St. Louis, MO; 1:200); rabbit anti-desmin (Abcam; 1:200); mouse anti-CD31 (Abcam; 1:25). The sections were then washed three times in TBS containing 0.1% Tween 20 and incubated with AlexaFluor 488 or AlexaFluor 568-conjugated secondary antibodies (Life Technologies, Carlsbad, CA) diluted 1:200 in TBS. The sections were then washed three times in TBS-0.1% Tween 20, rinsed with distilled water, and mounted with mounting medium containing DAPI (Vector Labs, Burlingame, CA).

Preparation of constructs for cryosectioning

The liver construct was rinsed once with DPBS, immersed in Tissue-Tek OCT compound (Sakura Finetek Europe B.V., Netherlands), and snap frozen. The frozen block containing the liver construct was then cut into 5 μm slices on a cryostat (Leica Cryocut 1800, Leica Microsystems). The slices were quickly fixed in -20 ° C liquid acetone and allowed to air dry for 20 minutes at room temperature. For Oil Red O staining, the slides were rehydrated in distilled water and immersed in 60% isopropanol for 2 minutes. At room temperature, the slides were stained with 0.3% w / v Oil Red O (Sigma-Aldrich) in 60% isopropanol for 15 minutes and subsequently rinsed with 60% isopropanol for 1 minute. The slides were briefly immersed in Gill's hematoxylin for counterstaining. The slides were rinsed with distilled water and fixed with an aqueous medium (American MasterTech).

Microscopic examination

H&E, PAS, and Oil Red O stained slides were imaged using a Zeiss Axioskop microscope (Zeiss, Jena, Germany). Images were acquired with an Insight2 camera and Spot 5.0 software (Diagnostic Instruments, Inc., Sterling Heights, MI). Fluorescently stained slides were imaged using a Zeiss AxioImager microscope and images were acquired with a Zeiss ICM-1 camera and Zen Pro software.

Preparation of S9 section

The medium was aspirated from the three-dimensional liver construct and 400 μl of phosphate buffered saline (Corning) supplemented with 1X protease inhibitor cocktail (Sigma) was added to the construct and subsequently frozen at -80°C. The construct was thawed and homogenized using a TissuRuptor (Qiagen) and disposable tip at 3000 rpm for 90 seconds, followed by a 90-second pause and then a 90-second homogenization at 3000 rpm. The homogenized supernatant was centrifuged at 4°C, 20,000 × g for 20 minutes. The supernatant was transferred to a new tube and subsequently subjected to protein quantification.

Protein quantification

Protein quantification was performed using the Bradford assay (Sigma) with bovine albumin as the standard (Sigma).

CYP 3A4 analysis

The S9 fraction was normalized to protein content and used in a Luciferin-IPA assay (CYP ProGlo; Promega) according to the manufacturer's protocol, and luminescence was read using a plate reader (Polarstar, BMG).

statistics

Statistical analysis was performed using ANOVA and students' t-test using GraphPad Prism software.

result

The bioprinted liver tissue contains a multicellular structure similar to human tissue. Like natural liver tissue, the bioprinted liver tissue contains specific cell types (e.g., hepatic stellate cells, hepatocytes, endothelial cells, etc.) enriched in specific areas, resulting in a patterned, compartmentalized structure.

The histological characterization of the liver tissue of bioprinting shows the key features of natural liver, including high cell density, and the production of ECM, lipids and glycogen. See Figure 21 (7th day after printing). The liver tissue of three-dimensional bioprinting shows striking histological features, including tight junctions (observed by visualization of E-cadherin, albumin and DAPI), and static astrocytes and microvascular structures (observed by visualization of desmin, CD31 and DAPI). The liver tissue of three-dimensional bioprinting shows compartmentalization that continues well after initial manufacturing. On the 21st day after printing, the liver cell area surrounded by non-substantial "interstitial" was determined. See Figure 22. Unexpectedly, the liver tissue of this bioprinting shows obvious self-organization into lobular structure, which simulates the natural liver very closely.

The observed structures supported function and viability for more than 40 days, enabling long-term testing, e.g., low doses, repeated exposures, and chronic pathological conditions. In 3D bioprinted liver tissue (primary hepatocytes), liver-specific function was maintained for more than 40 days. Bioprinted liver tissue showed continuous production of albumin for up to 6 weeks (42 days), far exceeding the time course of primary hepatocytes in published traditional 2D techniques. See Figure 23. Bioprinted liver tissue also showed continuous production of other hepatocyte factors such as cholesterol. See Figure 23.

3D bioprinted human liver tissue demonstrated the production of other relevant metabolites, including transferrin and fibrinogen, that were not achievable using existing 2D methods (see Figure 24).

The 3D bioprinted human liver also exhibited markers of bile canaliculi formation. Bile salt exporter (BSEP) is a membrane protein located in the canalicular membrane of hepatocytes. The main function of BSEP is to remove unconjugated and conjugated bile acids and salts from hepatocytes. In addition, BSEP expression indicates polarity within native tissues. Histological visualization of BSEP (IHC staining for BSEP protein), albumin, and DAPI showed that BSEP is present in hepatocyte albumin-producing cells. Figure 25 shows the calculation of the relative amount of BSEP mRNA relative to housekeeping gene RNA (18s RNA) in two bioprinted liver tissues 10 days after printing. The detectable levels of BSEP support the histological findings.

Compared to 2D primary hepatocytes, which lose function after a few days, bioprinted 3D liver tissue constructs showed sustained inducibility of active CYP3A4 enzyme (inhibitable by verapamil). See Figure 26.

3D bioprinted liver tissue showed a natural-like response to known hepatotoxicants, including diclofenac, trovafloxacin, and methotrexate. Figure 27 shows the induction of CYP3A4 at moderate doses of diclofenac, followed by damage at higher doses, as measured by lactate dehydrogenase (LDH). Figure 28 shows the reduction in albumin and increase in LDH observed in a multiple-dose study of trovafloxacin relative to levofloxacin (n=3, 2 doses in 4 days). The effect of trovafloxacin was observed to be closer to the clinical _Cmax (approximately 4 μM). This is in contrast to traditional two-dimensional primary hepatocyte cultures in which no LDH induction was observed. Figure 29 shows the reduction in viability (measured by ATP-cell titer glo) and albumin and increase in LDH observed in a multiple-dose study of methotrexate (n=3, daily dosing for 7 days).

3D bioprinted liver tissue showed a natural-like response to physiologically relevant molecules such as vitamin D. Vitamin D is important for maintaining homeostasis in the body. Vitamin D can be obtained through dietary intake or synthesized by cells in the skin. The liver processes vitamin D into metabolites that are important for bone formation/maintenance and into a form that can be excreted by the kidneys. Vitamin D also stimulates the expression of CYP3A4 in the liver and can potentially affect the bioavailability of drugs. Transcriptional profiling of bioprinted 3D liver tissue treated with 1, 5, and 10 μM vitamin D was performed. The relative expression of CYP3A4 transcripts was measured. Figure 30 shows that CYP3A4 activity was stimulated by approximately 7-fold and 4-fold for 5 and 10 μM vitamin D treatment, respectively.

Example 7 - Bioprinting of functional liver tissue with Kupffer cell incorporation

The introduction of Kupffer cells enabled the study of immunological aspects of Organovo's 3D liver tissue. Kupffer cells were introduced into the bioprinted liver construct at physiologically relevant percentages. The construct responded to LPS stimulation in a dose-dependent manner (LPS: Escherichia coli 0127:B8; Sigma). See Figure 31. Kupffer cells were bioprinted into the bioprinted liver construct at physiologically relevant percentages (approximately 12%). The construct responded to LPS stimulation in a dose-dependent manner. IL-10 and TNF-α are opposing cytokines. Expression of IL-10 reduced expression of TNF-α. This was observed within the bioprinted 3D liver tissue and was most pronounced at a dose of 100 μg/ml of LPS. See Figure 32.

Example 8 - Bioprinting of functional liver tissue with introduction of sinusoidal endothelial cells

The introduction of sinusoidal endothelial cells enables the study of immunological aspects of three-dimensional bioprinted liver tissue. Sinusoidal endothelial cells (HSEC) are a population of natural liver endothelial cells. Figure 33 shows a two-dimensional monolayer of HSEC. These cells have unique characteristics, including cell signaling for immunological responses. HSEC responds to LPS by producing IL-6 in 2D culture, as shown in Figure 34. In contrast, when HUVEC is stimulated by LPS, it does not produce detectable amounts of IL-6. When introduced into the bioprinting process, HSEC produce fused 3D liver tissue. Figure 35 shows fused three-dimensional liver tissue containing HSEC. To produce this tissue, sinusoidal endothelial cells are introduced into the non-parenchymal part to produce a fused structure.

Example 9 - Small molecule penetration

Experiments were conducted to determine whether small molecules could penetrate through 3D bioprinted liver tissue. 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate was supplemented at 1 μM to the culture medium of the 3D liver tissue for 24 hours. The construct was rinsed with 1 ml of HBSS buffer and the wash buffer was aspirated. This process was repeated three times. OCT embedding solution was added, and the construct was snap-frozen and cryosectioned. Five-micron sections were visualized using the FITC channel excited at 495 nm. The fluorescent component of the molecule 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate is excited at this wavelength. The section was sliced approximately 150 microns into the construct, and the fluorescently labeled compound was observed everywhere. The molecular weight of 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate is 529.28. Figure 36 shows FITC imaging of a cryosectioned 3D liver tissue at 5X, demonstrating small molecule penetration.

Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Various variations, modifications, and substitutions will now occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein are optionally used in practicing the present invention.

Claims (24)

1. An engineered, living three-dimensional liver tissue construct comprising at least one compartment defining a planar geometry, the compartment comprising an interior defined by a border, the interior comprising parenchymal cells, the parenchymal cells being hepatocytes or hepatocyte-like cells, the border comprising non-parenchymal cells; said cells aggregate to form a living three-dimensional liver tissue construct; provided that at least a portion of the construct is bioprinted and the construct is substantially free of pre-formed scaffolds when in use. 2. The construct according to claim 1, wherein the parenchymal cells are derived from one or more of the following sources: adult mammalian liver tissue; fetal mammalian liver tissue; induced pluripotent stem cells (iPSCs); iPSC-derived hepatocyte-like cells; adult stem cells/progenitor cells derived from the liver; and adult stem cells/progenitor cells derived from non-liver tissues. 3. The construct according to claim 1, wherein the non-solid cells include one or more of the following: vascular cells, endothelial cells, fibroblasts, mesenchymal cells, immune cells, Kupffer cells, astrocytes, bile duct epithelial cells, bile duct epithelioid cells, sinusoidal endothelial cells, liver-derived stem cells/progenitor cells, and non-liver-derived stem cells/progenitor cells. 4. The construct according to claim 1, comprising multiple layers. 5. The construct of claim 4, wherein at least one layer differs in composition or structure from at least one other layer to create a layered geometry. 6. The construct according to claim 1, used to enhance one or more liver functions in the human body. 7. The construct according to claim 6, for implantation in a subject at a site of injury, lesion, or degeneration. 8. An array comprising a plurality of engineered, living three-dimensional liver tissue constructs as described in claim 1, the array being used for in vitro analysis. 9. The array according to claim 8, used for one or more of the following: drug discovery; drug testing; preclinical studies; toxicity testing; absorption, distribution, metabolism, and excretion assays (ADME); drug metabolism and pharmacokinetics assays (DMPK); disease modeling; infectious disease modeling; host disease modeling; three-dimensional biological studies; and cell-based screening. 10. An engineered, living three-dimensional liver tissue construct comprising: one or more layers, each layer containing one or more hepatocyte types, the one or more layers agglomerating to form a living three-dimensional liver tissue construct, the construct characterized by having at least one of the following: a. At least one layer containing multiple cell types, which are spatially arranged relative to each other to create a planar geometry; and b. Multiple layers, at least one of which differs in composition or structure from at least one other layer, to create a layered geometry. The said construct includes at least one compartment with a defined planar geometry, the compartment having an interior defined by a side, the interior containing parenchymal cells, the parenchymal cells being hepatocytes or hepatocyte-like cells, and the side containing non-parenchymal cells. 11. The construct of claim 10, wherein at least one portion of the construct is bioprinted. 12. The construct according to claim 10, wherein the construct substantially does not contain a pre-formed scaffold when in use. 13. The construct of claim 10, wherein the parenchymal cells are derived from one or more of the following sources: adult mammalian liver tissue; fetal mammalian liver tissue; induced pluripotent stem cells (iPSCs); iPSC-derived hepatocyte-like cells; adult stem cells/progenitor cells derived from the liver; and adult stem cells/progenitor cells derived from non-liver tissues. 14. The construct of claim 10, wherein the non-solid cells comprise one or more of the following cell types: vascular cells, endothelial cells, fibroblasts, mesenchymal cells, immune cells, cancer cells, Kupffer cells, astrocytes, bile duct cells, sinusoidal endothelial cells, liver-derived stem cells/progenitor cells, and non-liver-derived stem cells/progenitor cells. 15. The construct according to claim 10, used for enhancing one or more liver functions in the human body. 16. The construct of claim 15, for implantation in a subject at a site of injury, lesion, or degeneration. 17. An array comprising a plurality of engineered, living three-dimensional liver tissue constructs according to claim 10, the array being used for in vitro analysis. 18. The array of claim 17, used for one or more of the following: drug discovery; drug testing; preclinical studies; toxicity testing; absorption, distribution, metabolism, and excretion assays (ADME); drug metabolism and pharmacokinetics assays (DMPK); disease modeling; infectious disease modeling; host disease modeling; three-dimensional biological studies; and cell-based screening. 19. A method for manufacturing a living three-dimensional liver tissue construct, comprising: a. To prepare one or more types of bioinks containing non-celled organisms; b. Prepare one or more bioinks containing parenchymal cells, wherein the parenchymal cells are hepatocytes or hepatocyte-like cells; c. Deposit the bio-ink onto the support; and d. Incubate the deposited bioink for approximately 1 hour to approximately 30 days to form a living three-dimensional liver tissue construct comprising at least one compartment with a defined planar geometry, the compartment comprising an interior containing parenchymal cells and defined by a border containing non-parenchymal cells. 20. The method of claim 19, wherein the non-solid cells comprise one or more of the following: vascular cells, endothelial cells, fibroblasts, mesenchymal cells, immune cells, cancer cells, Kupffer cells, astrocytes, bile duct epithelial cells, bile duct epithelioid cells, sinusoidal endothelial cells, liver-derived stem cells/progenitor cells, and non-liver-derived stem cells/progenitor cells. 21. The method of claim 19, wherein the parenchymal cells are derived from one or more of the following sources: adult mammalian liver tissue; fetal mammalian liver tissue; induced pluripotent stem cells (iPSCs); iPSC-derived hepatocyte-like cells; adult stem cells/progenitor cells derived from the liver; and adult stem cells/progenitor cells derived from non-liver tissues. 22. The method of claim 19, wherein at least one portion of the construct is bioprinted. 23. The method of claim 19, wherein the construct contains substantially no pre-formed scaffold when in use. 24. The method of claim 19, wherein the construct is not neurally innervated.