3D bioprinted liver

Abstract

The present disclosure provides 3D bio-printed livers and methods of making or using the same. Aspects of the disclosure relate to compositons of 3D bio-printed livers. In some embodiments, the 3D bio-printed livers comprise a mesh casing, a plurlaity of microporous organoid-infused microgel matrices, and a hetaptic vasculature. Other aspects of the disclosure relate to methods of making the 3D bio-printed livers dislcosed herein. In some embodiments, the methods relate to creating a mesh casing in the shape of a liver, or portion thereof, loading a plurality of microporous organoid-infused microgel matrices into the mesh casing, and using embedded bioprinting to create a hepatic artery, a portal vein, and biliary duct within the mesh casing.

Description

[0001] 3D BIOPRINTED LIVER

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 686,458, filed August 23, 2024, entitled “3D Bioprinted Liver,” incorporated herein by reference in its entirety.

[0004] FIELD

[0005] The present disclosure generally relates to three-dimensional bio-printed organs, e.g., livers, and methods of making and using such organs.

[0006] BACKGROUND

[0007] Organ shortage is a worldwide crisis affecting millions of patients who need transplants. Despite efforts to promote organ donation, and advancements in technology and therapies to increase the viability of organ transplants, there is an urgent need to provide alternatives to tackle organ shortage and transplant rejection effectively.

[0008] It has been known for thousands of years that liver can regenerate. However, despite its regenerative ability, liver failure is a devastating condition that results in jaundice and death. Accordingly, improvements in the field of liver organ transplantation are needed.

[0009] SUMMARY

[0010] The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and / or a plurality of different uses of one or more systems and / or articles.

[0011] In some aspects, the present disclosure relates to a 3D bio-printed liver, including: a porous cell bed; and a hepatic vasculature contained within the porous cell bed.

[0012] In some aspects, the present disclosure relates to a 3D bio-printed organ, including: A porous cell bed in a shape of a target organ, or fragment thereof; and a vasculature contained within the porous cell bed.

[0013] In some aspects, the present disclosure relates to a method for producing a 3D bioprinted liver, or portion thereof, including: creating a porous cell bed in a shape of liver, or portion thereof; and using embedded bioprinting to create a hepatic artery, a portal vein, and biliary duct within a mesh casing.

[0014] In some aspects, the present disclosure relates to a method for producing a 3D bioprinted organ, or portion thereof, including: creating a porous cell bed in a shape of liver, or portion thereof; and using embedded bioprinting to create an organ vasculature within the porous cell bed. In some aspects, the present disclosure relates to a 3D bio-printed liver, including: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a hepatic vasculature within the mesh casing.

[0015] In some aspects, the present disclosure relates to a method for producing a 3D bioprinted liver, including: creating a mesh casing in a shape of a liver, or portion thereof; loading a plurality of microporous organoid-infused microgel matrices into the mesh casing; and using embedded bioprinting to create a hepatic artery, a portal vein, and biliary duct within the mesh casing.

[0016] In some aspects, the present disclosure relates to a 3D bio-printed organ, including: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a vasculature within the mesh casing.

[0017] In some aspects, the present disclosure relates to a method for producing a 3D bioprinted organ, including: creating a mesh casing in a shape of an organ, or portion thereof; loading a plurality of microporous organoid-infused microgel matrices into the mesh casing; and using embedded bioprinting to create an organ vasculature within the mesh casing.

[0018] In some aspects, the present disclosure relates to a 3D bio-printed organ, including: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a vascular network, wherein a portion of the vascular network includes a plurality of channels having an outer diameter (OD) greater than 2 mm.

[0019] In some aspects, the present disclosure relates to a 3D bio-printed organ, including: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a vascular network, wherein a portion of the vascular network includes a plurality of channels having an OD of between 700 microns and 2 mm.

[0020] In some aspects, the present disclosure relates to a 3D bio-printed organ, including: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a vascular network, wherein a portion of the vascular network includes a plurality of channels having an OD of between 5 microns and 50 microns.

[0021] In some aspects, the present disclosure relates to a 3D bio-printed organ, including: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a vascular network, wherein the vascular network includes: (i) a first plurality of channels having an outer diameter (OD) of greater than 2 mm, and (ii) a third plurality of channels having an OD of between 5 microns and 50 microns.

[0022] In some aspects, the present disclosure relates to a 3D bio-printed organ, including: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a vascular network, wherein the vascular network includes: (i) a second plurality of channels having an outer diameter (OD) of between 700 microns and 2 mm, and (ii) a third plurality of channels having an OD of between 5 microns and 50 microns.

[0023] In some aspects, the present disclosure relates to a 3D bio-printed organ, including: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a vascular network, wherein the vascular network includes: (i) a first plurality of channels having an outer diameter (OD) of greater than 2 mm, and (ii) a second plurality of channels having an OD of between 700 microns and 2 mm.

[0024] In some aspects, the present disclosure relates to a 3D bio-printed organ, including: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a vascular network, wherein the vascular network includes: (i) a first plurality of channels having an outer diameter (OD) of greater than 2 mm, (ii) a second plurality of channels having an OD of between 700 nm and 2 mm, and (iii) a third plurality of channels having an OD of between 5 microns and 50 microns.

[0025] In some aspects, the present disclosure relates to a method for producing a 3D bioprinted organ, including: creating a mesh casing in a shape of a target organ, or portion thereof; loading a plurality of microporous organoid-infused microgel matrices into the mesh casing; and using embedded bioprinting to create a vascular network including a plurality of channels having an OD of greater than 2 mm.

[0026] In some aspects, the present disclosure relates to a method for producing a 3D bioprinted organ, including: creating a mesh casing in a shape of a target organ, or portion thereof; loading a plurality of microporous organoid-infused microgel matrices into the mesh casing; and using embedded bioprinting to create a vascular network including a plurality of channels having an OD of between 700 microns and 2 mm.

[0027] In some aspects, the present disclosure relates to a method for producing a 3D bioprinted organ, including: creating a mesh casing in a shape of a target organ, or portion thereof; loading a plurality of microporous organoid-infused microgel matrices into the mesh casing; and using embedded bioprinting to create a vascular network including a plurality of channels having an OD of between 5 microns and 50 microns.

[0028] In some aspects, the present disclosure relates to a method for producing a 3D bioprinted organ, including: creating a mesh casing in a shape of a target organ, or portion thereof; loading a plurality of microporous organoid-infused microgel matrices into the mesh casing; and using embedded bioprinting to create a vascular network, wherein the vascular network includes: (i) a first a plurality of channels having an OD of between 5 microns and 50 microns, and (ii) a third plurality of channels having an OD of between 5 microns and 50 microns.

[0029] In some aspects, the present disclosure relates to a method for producing a 3D bioprinted organ, including: creating a mesh casing in a shape of a target organ, or portion thereof; loading a plurality of microporous organoid-infused microgel matrices into the mesh casing; and using embedded bioprinting to create a vascular network, wherein the vascular network includes: (i) a second plurality of channels having an outer diameter (OD) of between 700 microns and 2 mm, and (ii) a third plurality of channels having an OD of between 5 microns and 50 microns.

[0030] In some aspects, the present disclosure relates to a method for producing a 3D bioprinted organ, including: creating a mesh casing in a shape of a target organ, or portion thereof; loading a plurality of microporous organoid-infused microgel matrices into the mesh casing; and using embedded bioprinting to create a vascular network, wherein the vascular network includes: (i) a first plurality of channels having an outer diameter (OD) of greater than 2 mm, and (ii) a second plurality of channels having an OD of between 700 microns and 2 mm.

[0031] In some aspects, the present disclosure relates to a method for producing a 3D bioprinted organ, including: creating a mesh casing in a shape of a target organ, or portion thereof; loading a plurality of microporous organoid-infused microgel matrices into the mesh casing; and using embedded bioprinting to create a vascular network, wherein the vascular network includes: (i) a first plurality of channels having an outer diameter (OD) of greater than 2 mm, (ii) a second plurality of channels having an OD of between 700 nm and 2 mm, and (iii) a third plurality of channels having an OD of between 5 microns and 50 microns.

[0032] Several methods are disclosed herein of administering a subject with a compound for prevention or treatment of a particular condition. It is to be understood that in each such aspect of the disclosure, the disclosure specifically includes, also, the compound for use in the treatment or prevention of that particular condition, as well as use of the compound for the manufacture of a medicament for the treatment or prevention of that particular condition.

[0033] In another aspect, the present disclosure encompasses methods of making one or more of the embodiments described herein, for example, a 3D bio-printed liver. In still another aspect, the present disclosure encompasses methods of using one or more of the embodiments described herein, for example, a 3D bio-printed liver. Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the accompanying figures.

[0034] BRIEF DESCRIPTION OF DRAWINGS

[0035] Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. In the figures:

[0036] Figs. 1A-1D show a series of schematics illustrating universal liver bioprinting, as contemplated herein. Fig. 1 A shows GMP level manufacturing of liver organoid microgel matrix with hypo-immune liver cells packing on a suturable PGS case followed by annealing, according to some embodiments. Fig. IB shows embedded bioprinting of a vascular network in the microporous organoid-infused microgel matrix via robotic bioprinting, according to some embodiments. Fig. 1C shows a schematic of a cross-section of microporous organoid- infused microgel matrix before printing, according to some embodiments. Fig. ID shows a schematic of a cross-section of microporous organoid-infused microgel matrix after printing and maturation, according to some embodiments.

[0037] Figs. 2A-2B shows plots of the hypo-immunogenicity of B2M- / -, HLA-E+ 19-9-11 iPSCs. WT, B2M7', and B2M_ / 7HLA-E+iPSCs co-cultured with human T cells (Fig. 2A) or NK cells (Fig. 2B), according to some embodiments.

[0038] Fig. 3 illustrates the directed differentiation of iPSCs to AFP+hepatocytes, CK19+cholangiocytes, PDGFR+stellate cells, CD206+Kupffer cells, and CD31+ECs, according to some embodiments.

[0039] Figs. 4A-4H illustrate the derivation of liver microgels according to some embodiments. FIG. 4A shows a photomicrograph of a liver microgel on Day 9. Fig. 4B shows a fluorescent photomicrograph of the microtissue structure of liver microgel stained with live / dead cell dyes. Fig. 4C shows a fluorescent photomicrograph of bile canaliculi formation in liver microgel as indicated by CDFDA staining. Fig. 4D shows a photomicrograph of Ki67 IHC staining of liver microgel. Red arrows indicate the proliferating hepatocytes. Fig. 4E shows a photomicrograph of H&E staining of liver microgel. Figs. 4F-4H show plots of the viability of hepatocytes culture in 2D or microgel at different densities (Fig. 4F), the productions of albumin (Fig. 4G), and the productions of urea in 2D and liver microgels (Fig. 4H), according to some embodiments.

[0040] Figs. 5A-5E illustrate the development of an exemplary perfusable vascularized multicellular liver platform, according to some embodiments. Fig. 5 A shows a fluorescent photomicrograph of devices with control ECs or reprogrammed ECs (R-VECs) stained with human VEcad at day 7. Scale bars, 3 mm. Fig. 5B shows fluorescent photomicrographs of R- VECs co-assembled with hepatocytes and stellate cells to form perfused tissues. Fig. 5C shows optical and fluorescent photomicrographs of constructs with channels and microvasculature, vessel sprouts and anastomoses. Fig. 5D shows a high magnification fluorescent photomicrograph of the anastomosis. Fig. 5E shows a schematic of the hierarchical vasculature, according to some embodiments.

[0041] Figs. 6A-6C illustrate an exemplary 3D embedded-bioprinting approach, as contemplated herein (Fig. 6A). Fig. 6B shows a fluorescent photomicrograph of a 3D branch structure formed via embedded printing in a hydrogel. Fig. 1C shows fluorescent photomicrographs of ECs forming a monolayer inside hydrogel bio-printed microchannels, according to some embodiments.

[0042] Fig. 7 illustrates an exemplary microfluidic device for the large scale production of organoid microgels. Fig. 7A shows an exemplary 3D printed parallel microfluidic device used in the production of microgels at 500 mL / hr. The exemplary device shown has 16 parallel channels. Fig. 7B shows the flow of the precursor solution and droplet formation within the device. Fig. 7C shows the accumulation of microgels produced using the exemplary device.

[0043] Fig. 8 illustrates an exemplary workflow for bioprinting vascular networks within a casing comprising a plurality of organoid microgels. The exemplary workflow shown comprises obtaining a CAD model of human liver from a CT scan. The CAD model is then sliced in a slicer software to calculate its layer-by-layer coordinates. The two-part shell model of liver geometry is then used in the robotic embedded bioprinting software to print the vascular network.

[0044] DETAILED DESCRIPTION

[0045] The present disclosure provides 3D bio-printed livers or other organs, and methods of making or using the same. Aspects of the disclosure relate to compositions of 3D bio-printed livers or other organs,. In some embodiments, the 3D bio-printed livers comprise a mesh casing, a plurality of microporous organoid- infused microgel matrices, and a hetaptic vasculature. Other aspects of the disclosure relate to methods of making the 3D bio-printed livers or other organs dislcosed herein. In some embodiments, the methods relate to creating a mesh casing in the shape of a liver or other organ, or portion thereof, loading a plurality of microporous organoid-infused microgel matrices into the mesh casing, and using embedded bioprinting to create a hepatic artery, a portal vein, and biliary duct within the mesh casing.

[0046] Some embodiments are directed to functional, scalable, and / or transplantable bioprinted livers or other organs. Some embodiments contemplate making cost effective organs by: 1) using allogeneic hypoimmunogenic iPSC (hiPSC)-derived liver cells that can be used as a ‘universal cell’ that avoid expensive patient-specific cell derivation; 2) using media by comparing existing serum-free media formulations for iPSC growth and differentiation with a bovine platelet lysate (bPL) product. In this process, through weekly bPL collections through apheresis about -500 L of plasma can be collected from a single cow annually (compared to 300 mL from a fetal cow) without health or welfare concerns. See, e.g., US Pat. Apl. Pub. No. 2024 / 0150724, or Int. Pat. Apl. Pub. No. WO 2022 / 192446, each incorporated herein by reference in its entirety.

[0047] To bio-print livers or or other organs that can be sutured, some of the bio-printed livers or other organs contemplated herein may comprise a long-term biodegradable flexible casing with suturable ports, comprising poly(glycerol sebacate) (PGS), a biorubber that is biocompatible and suturable. In some embodiments, the casing may comprise PGS meshes that are weaved into liver shape. In some embodiments, the casing comprises one or more openings. In some embodiments, the casing comprises an open top to allow for cell deposition and embedded bioprinting. Additionally, in some embodiments, the casing may then be closed aseptically with the ports easily accessible for anastomosis and implantation (Fig. 1A).

[0048] In some embodiments, a bio-printed liver or other organs comprises one or more micro-architecture polymeric (MAP) micro-annealed gels. Compared to solid gels, within which only diffusion occurs, MAP micro-annealed gels may provide better control over tissue mechanics, porosity, convection and customization. Without wishing to be bound by any particular theory, it is believed that liver organoids can be encapsulated within MAPs, which can then be packed into porous structures with the inter-MAP space enabling perfusion and vascular tissue formation. hiPSC-derived liver organoids will be encapsulated in microgels containing ECMs that are known in the art to enhance liver cell maturation. In some embodiments, the casing is filled with liver organoid MAP gels.

[0049] In some embodiments, embedded bioprinting may be used to generate larger vessels and bile networks. Embedded bioprinting is a technique in which sacrificial materials and cells can be printed within a supportive matrix (e.g., MAP annealed gels containing organoids) that provides structural support during the printing process. In some embodiments, embedded bioprinters are used to generate distinct channel networks (e.g., inputs for portal vein and hepatic artery and outputs for bile duct and hepatic vein), for example, by using alginate and Pluronic bioinks (Fig. IB). Those of skill in the art will understand and appreciate, however, that the invention is not limited to only alginate and Pluronic bioinks, and any suitable bioink may be used herein. In some embodiments, the printed networks connect the input and output ports to the microporous annealed organoids. In some embodiments, the channels are lined with ECs and cholangiocytes, for example, as needed to support the maturation process (Fig. 1C).

[0050] In some embodiments, a commercial organ perfusion bioreactors (e.g., Harvard Apparatus for mouse size livers and TransMedics for human size bio-printed livers) may be used to generate pulsatile flow mimicking in vivo hemodynamics to mature the tissues (Fig. ID). In some embodiments, the tissues may further mature in vivo.

[0051] In some embodiments, the 3D bio-printed livers or other organs disclosed herein comprise a mesh casing, a plurality of microporous organoid-infused microgel matrices, and a hepatic vasculature. In some embodiments, the mesh casing comprises poly(glycerol sebacate). In some embodiments, the mesh casing comprises an open top. In some embodiments, the mesh casing comprises one or more suturable ports (e.g., for suturing to existing tissue). In some embodiments, a plurality of microporous organoid-infused microgel matrices comprises one or more micro-architectured polymeric (MAP) micro-annealed gels. In some embodiments, the one or more MAP micro-annealed gels comprise one or more hiPSC-derived liver organoids. In some embodiments, the hepatic vasculature comprises a hepatic artery. In some embodiments, the hepatic vasculature comprises a portal vein. In some embodiments, the hepatic vasculature comprises a bile duct. In some embodiments, the hepatic artery, portal vein, and the bile duct are parallel to each other.

[0052] Other aspects relate to one or more methods. In some embodiments, the methods relate to producing a 3D bio-printed liver or other organ. In some embodiments, the methods comprise creating a mesh casing in the shape of a liver or other organ, or portion thereof, loading a plurality of microporous organoid-infused microgel matrices into the mesh casing, and using embedded bioprinting to create a hepatic artery, a portal vein, and biliary duct within the mesh casing. In some embodiments, the methods further comprise maturing the 3D bio-printed liver or other organ in a bioreactor. In some embodiments, the methods further comprise endothelializing the hepatic artery and portal vein with ECs. In some embodiments, the methods further comprise endothelializing the biliary duct with cholangiocytes.

[0053] In addition, certain embodiments are directed to using organs such as those described herein. For example, an organ such as a 3D bio-printed liver may be transplanted into a subject, e.g., in need thereof. The subject may be human, or a non-human animal. Examples of subjects include, but are not limited to, a mammal such as a cow, sheep, goat, horse, rabbit, pig, mouse, rat, dog, cat, a primate (e.g., a monkey, a chimpanzee, etc.), or the like. In some cases, the subject is a non-mammal such as a bird, an amphibian, or a fish. In some embodiments, the subject is genetically engineered.

[0054] In addition, some aspects of the present disclosure relate to 3D bio-printed livers or other organs. In some embodiments, a 3D bio-printed liver, or other organ, comprises a porous cell bed. A porous cell bed includes a porous substrate capable of supporting cell growth in certain embodiments. In some embodiments, a porous cell bed may have discrete pores, interconnected pores, or a combination thereof. In some embodiments, the porous substrate is biocompatible. In some embodiments, the porous substrate comprises one or more surface topographies, e.g., that influence cell attachment, orientation, and / or function. In some embodiments, the porous substrate comprises one or more specific mechanical properties, e.g., that influence cell attachment, motility, and / or differentiation.

[0055] Accordingly, any suitable material known to the skilled artisan capable of satisfying the above criteria may be used as a porous cell bed in the instant disclosure. For example, in some embodiments, the porous cell bed comprises a porous polymeric scaffold, a hydrogel scaffold, a porous ceramic scaffold, a decellularized natural tissue, or a combination thereof. Exemplary embodiments of possible polymeric scaffolds include, but are not limited to, scaffold comprising poly (glycerol sebacate) (PGS), polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), poly(lactic-co-glycolic acid) (PLGA), polhydroxyalkanoates (PHAs), poly(trimethylene carbonate), poly(ethylene succinate), and PGS, or a combination thereof. Exemplary embodiments of hydrogel scaffolds include, but are not limited to silk fibroin, collagen, chitosan, gelatin, fibrin, hyaluronic acid (HA) agarose, and alginate. Exemplary embodiments, of porous ceramic scaffolds include, but are not limited to, hydroxyapatite (HA), tricalcium phosphate (TCP), biphasic calcium phosphate (BCP), bioactive glass, zirconia (ZrO2), silicon nitride (SisN4), calcium silicate (CaSiOa), alumina (AI2O3), forsterite (Mg2SiO4), and Wollastonite (CaSiOa). Exemplary embodiments of decellularized natural tissues include, but are not limited to, lung, intestine, plant leaves, etc.). In some embodiments, a porous cell bed comprises a plurality of organoids encapsulated with hydrogel microparticles (e.g., organoid loaded microgels). In some cases, the 3D bio-printed livers or other organs comprise a casing or mold to hold the plurality of organoid loaded microgels. In some embodiments, the casing is flexible. In some embodiments, the casing is biodegradable. In some embodiments, the casing comprising one or more suturable ports. In some embodiments, the casing comprises an open top to allow for cell deposition and embedded bioprinting. Additionally, in some embodiments, the casing may then be closed aseptically with the ports easily accessible for anastomosis and implantation (Fig. 1A).

[0056] The casing may be made of any suitable material known to the skilled artisan. In some embodiments, the casing comprises poly (glycerol sebacate) (PGS), a biorubber that is biocompatible and suturable. The skilled artisan will understand that the casing may comprise materials other than PGS. For example, in some embodiments, the casing comprises silk fibroin, collagen, chitosan, alginate, polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), poly(lactic-co-glycolic acid) (PLGA), polhydroxyalkanoates (PHAs), poly(trimethylene carbonate), poly(ethylene succinate), and PGS, or a combination thereof.

[0057] In some embodiments, the casing is woven into a mesh-like structure. The casing may be woven using any suitable technique known to the skilled artisan. For example, in some embodiments, the casing is woven via electrospinning, melt spinning, wet spinning, dry spinning, textile weaving or knitting, braiding or filament winding.

[0058] In some embodiments, the casing is cut into a shape of a target organ (e.g., liver) or a portion thereof (e.g., a portion of a liver). For example, in some embodiments, the casing is cut into a shape of a liver, kidney, spleen, stomach, intestine, heart, brain, pancreas, or any portion thereof. Any technique known to the skilled artisan may be used to cut the casing into the desired shape. Exemplary embodiments include, but are not limited to, laser cutting, die cutting, plotter cutting (e.g., knife or blade), waterjet cutting, punching, or CNC milling.

[0059] In some embodiments, a casing comprises a coating. In some cases, the coating is an antifouling coating. In some embodiments, the coating comprises hyaluronic acid methacrylate (HAMA) and hydrosulfuryl heparin (HepSH). However, any suitable material known to the skilled artisan to be antifouling may be used to coat the casing. For example, in some embodiments, the antifouling coating comprises polyethylene glycol (PEG), poly(ethylene oxide) (PEG), zwitterionic polymers (e.g., poly (carboxybetaine)(PCB), poly(sulfobetaine)(PSB), or poly(trimethylamine N-oxide)(PTMAO)), polyglycerol, hyaluronic acid (HA), PEGylated polyurethanes, amphiphilic block copolymers (e.g., PEG-b- PDMS), polydopamine, PEGylated heparin, slippery liquid-infused porous surfaces (SLIPS), or any combination thereof.

[0060] A casing may be coated using any one of the coatings contemplated herein using any suitable technique known to the skilled artisan. Exemplary embodiments include, but are not limited to, chemical grafting (e.g., via click chemistry or other bioconjugation chemistry), plasma polymerization, self-assembled monolayers (SAMs), or layer-by-layer assembly.

[0061] In some embodiments, a 3D bio-printed livers or other organ, as disclosed herein, comprises a plurality of cells. In some embodiments, the cells are organized into a plurality of organoids. The organoid, in certain embodiments, may be a 3D cell culture that mimics the structure, function, and / or complexity of human organs. The cells used are often derived from stem cells, however, the skilled artisan will understand that this is not required, and any suitable cell type may be used. In some embodiments, human induced pluripotent stem cells (hiPSCs) are differentiated into a target cell type, which is subsequently used to create a target organoid. For example, in some embodiments, hiPSC cells are differentiated into neural progenitor cells, radial glia cells, cortical neurons, astrocytes, and / or oligodendrocytes, and at least one or more of said cells are used to create brain organoids.

[0062] In some embodiments, hiPSCs are differentiated into definitive endoderm, anterior foregut endoderm, lung progenitor cells, alveolar epithelial cells, and / or airway epithelial cells, and at least one or more of said cells are used to create lung and airway organoids.

[0063] In some embodiments, hiPSCs are differentiated into cardiac mesoderm cells, cardiomyocytes, cardiac fibroblasts, endothelial cells, smooth muscle cells, and at least a portion of said cells are used to create cardiac organoids.

[0064] In some embodiments, hiPSCs are differentiated into retinal progenitor cells, retinal pigment epithelium, and / or photoreceptor cells, and at least one or more of said cells are used to create retinal organoids.

[0065] In some embodiments, the hiPSCs are differentiated into otic placode-like progenitor cells and / or hair precursor cells, and at least one or more of said cells are used to create inner ear organoids.

[0066] In some embodiments, the hiPSCs are differentiated into definitive endoderm cells, gastric progenitor cells, intestinal stem cells, enterocytes, goblet cells, and / or Paneth cells, and at least one or more of said cells are used to create gastric organoids and / or intestinal organoids. In some embodiments, the hiPSCs are differentiated into hepatic endoderm cells, hepatocytes, and / or cholangiocyte, and at least one or more of said cells are used to create liver organoids.

[0067] In some embodiments, the hiPSCs are differentiated into pancreatic progenitor cells or endocrine cells, and at least one or more of said cells are used to create pancreatic organoids and / or islet- like structures.

[0068] In some embodiments, the hiPSCs are differentiated into intermediate mesoderm cells, nephron progenitors cells, podocytes, and / or proximal / distal tubule cells, and at least one or more of said cells are used to create kidney organoids.

[0069] In some embodiments, the hiPSCs are differentiated into hemogenic endothelium, hematopoietic progenitors, macrophages, and / or dendritic cells, and at least one or more of said cells are used to create bone marrow-like organoids, thymic organoids, and / or immune- organoid co-cultures.

[0070] In some embodiments, the hiPSCs are differentiated into surface ectoderm cells, keratinocytes, dermal fibroblasts, and / or melanocytes, and at least one or more of said cells are used to create skin organoids (e.g., with hair follicles) and / or epidermal organoids.

[0071] In some embodiments, the hiPSCs are differentiated into gonadal progenitor cells, Sertoli and Leydig-like cells (e.g., testis), and / or granulosa-like cells (e.g., ovaries), and at least one or more of said cells are used to create testicular organoids or ovarian organoids, respectively.

[0072] In some embodiments, the hiPSCs are hypoimmunogenic. In some embodiments, the hiPSCs are B2M- / -, CIITA- / -, and / or HLA-E+. In some embodiments, the hiPSCs comprise a Failsafe suicide gene switch. Those of skill in the art will understand that the invention is not limited to a Failsafe suicide gene switch and that any suitable suicide gene switch may be used (e.g., to preserve cell line integrity) in the hiPSCs contemplated herein.

[0073] Although hiPSCs) are described in certain embodiments herein, it will be understood by those skilled in the art that the invention is not limited to the use of hiPSCs. Any suitable cell type, including but not limited to embryonic stem cells (ESCs), adult stem cells, primary cells, progenitor cells, immortalized cell lines, or differentiated somatic cells, may be employed depending on the intended application.

[0074] In some embodiments, a 3D bio-printed liver or other organ, as disclosed herein, comprises a plurality of organoids encapsulated within a hydrogel microparticle, referred to herein as an “organoid microgel”. As used herein, the terms “organoid microgel” is synonymous with the terms “micro-architectured polymeric (MAP) micro-annealed gels” and “microporous organoid-infused microgel matrices.” In some embodiments, a casing comprises a plurality of organoid microgels (e.g., a plurality of organoid microgels are located within a casing). In some embodiments, a 3D bio-printed liver or other organ comprises one or more micro-architectured polymeric (MAP) microgels. Compared to solid gels, within which only diffusion occurs, MAP microgels may provide better control over tissue mechanics, porosity, convection and customization. Without wishing to be bound by any particular theory, it is believed that organoids (e.g., liver organoids) can be encapsulated within microgels, which can then be packed within a casing (e.g., biodegradable casing) to yield porous structures with the interstitial space between adjacent microgels enabling perfusion and vascular tissue formation.

[0075] In some embodiments, a 3D bio-printed liver, or other organ, comprises hiPSC- derived organoids (e.g., liver organoids, kidney organoids, heart organoids, etc.) encapsulated in microgels. The microgels, in some embodiments, comprise extracellular matrix proteins (ECMs) that are known in the art to enhance liver cell (or other cell) maturation. In some embodiments, the microgels comprises one or more polymers. In some embodiments, the one or more polymers comprise methacrylate gelatin, polyethylene glycol diacrylate, norbornene-functionalized polyethylene glycol, hyaluronic acid methacrylate, alginate, dextran methacrylate, fibrinogen methacrylate, silk fibroin methacrylate, and / or polyvinyl alcohol modified with acrylate or thiol group.

[0076] As mentioned above, in some embodiments, a 3D bio-printed organ comprises a plurality of organoid-infused microgels that have been annealed. The annealing process may include crosslinking at least a portion of the plurality of organoid-infused microgels to one another to produce a porous bed comprising a plurality of inter-particle crosslinks. Any suitable method known to the skilled artisan may be used to crosslink (e.g., anneal) the plurality of organoid-infused microgels disclosed herein. For example, in some embodiments, a crosslinking agent is used to anneal (e.g., crosslink) the organoid-infused microgels. Exemplary crosslinking agents include, but are not limited to, genipin, l-Ethyl-3- (3-dimethylaminopropyl)carbodiimide and N-Hydroxy succinimide)(EDC / NHS), glutaraldehyde, transglutaminase, riboflavin, thiol-Michael addition agents, photocrosslinking agents, enzymatic crosslinking agents, and tannic acid. In some embodiments, the crosslinking agent is genipin.

[0077] In some embodiments, the concentration of crosslinking agent is between 0.1 % and 1 % (wt / wt). In some embodiments, the concentration of crosslinking agent is greater than or equal to 0.1 % (wt / wt), greater than or equal to 0.2 % (wt / wt), greater than or equal to 0.3 % (wt / wt), greater than or equal to 0.4 % (wt / wt), greater than or equal to 0.5 % (wt / wt), greater than or equal to 0.6 % (wt / wt), greater than or equal to 0.7 % (wt / wt), greater than or equal to 0.8 % (wt / wt), greater than or equal to 0.9 % (wt / wt). In some embodiments, the concentration of crosslinking agent is of less than or equal to 1 % (wt / wt), less than or equal to 0.9 % (wt / wt), less than or equal to 0.8 % (wt / wt), less than or equal to 0.7 % (wt / wt), less than or equal to 0.6 % (wt / wt), less than or equal to 0.5 % (wt / wt), less than or equal to 0.4 % (wt / wt), less than or equal to 0.3 % (wt / wt), less than or equal to 0.2 % (wt / wt). Combinations are also possible in some embodiments. For example, in some embodiments, the concentration of crosslinking agent is greater than or equal to 0.1 % (wt / wt) and less than or equal to 1 % (wt / wt). Combinations of other ranges are also possible (e.g., greater than or equal to 0.1 % (wt / wt) and less than or equal to 1 % (wt / wt)). Other ranges are also possible.

[0078] In some embodiments, a 3D bio-printed liver or other organ comprises a plurality of channels (e.g., a vasculature) within a biodegradable flexible casing (e.g., a mesh casing). In some embodiments, the vasculature comprises a plurality of channels. In some embodiments, at least a portion of the channels have an outer diameter of between 0.005 mm and 50 mm. In some embodiments, at least a portion of the channels have an outer diameter of greater than or equal to 0.005 mm, greater than or equal to 10 mm, greater than or equal to 20 mm, greater than or equal to 30.005 mm or greater than or equal to 40 mm. In some embodiments, at least a portion of the channels have an outer diameter of less than or equal to 50 mm, less than or equal to 40 mm, less than or equal to 30 mm, less than or equal to 20 mm or less than or equal to 10 mm. Combinations are also possible in some embodiments. For example, in some embodiments, at least a portion of the channels have an outer diameter of greater than or equal to 0.005 mm and less than or equal to 50 mm. Combinations of other ranges are also possible (e.g., greater than or equal to 0.005 mm and less than or equal to 50. mm). Other ranges are also possible. For example, in some embodiments, at least a portion of the channels have an outer diameter of greater than 2 mm (OD). In some embodiments, at least a portion of the channels have an outer diameter of between 700 microns and 2 mm (OD).

[0079] In some embodiments, a plurality of channels (e.g., vasculature) within a 3D bioprinted organ (e.g. liver) are endothelialized with induced endothelial cells (iECs). In some embodiments, a plurality of channels (e.g., vasculature) within a 3D bio-printed organ (e.g. liver) are endothelialized with primary endothelial cells.

[0080] In some embodiments, a 3D bio-printed organ is a 3D bio-printed liver. In some embodiments, the 3D bio-printed liver comprises a hepatic vasculature. In some embodiments, the hepatic vasculature comprises a hepatic artery, a portal vein, and / or a bile duct. In some embodiments, the hepatic artery and / or the portal vein comprises iECs (e.g., are endothelialized with iECs). In some embodiments, the bile duct comprises induced cholangiocytes (iChols) (e.g., are endothelialized with iChols). In some embodiments, the hepatic artery, portal vein, and the bile duct are parallel to each other.

[0081] Other aspects relate to one or more methods. In some embodiments, the methods relate to producing a 3D bio-printed liver or other organ. In some embodiments, the methods comprise forming a porous cell bed. Any suitable method known to the skilled artisan for creating a porous cell bed may be used to produce the porous cell bed as disclosed herein. In some embodiments, the porous cell bed comprises a porous polymer scaffold, the porous polymeric scaffold formed via solvent casting, particulate leaching, gas foaming, electrospinning / electrospraying, and / or phase separation / freeze drying.

[0082] In some embodiments, the porous cell bed comprises hydrogels. While hydrogels are naturally porous at the angstrom length scale, there are many art recognized techniques to generate larger pores within hydrogel-based scaffolds, including for example, cryogelation (e.g., freeze a hydrogel precursor to form ice crystals, which melt during thawing and leave behind pores) and / or freeze-drying (e.g., creation of pores via subliming a frozen porogen). In other embodiments, a porous cell bed may be made via microgel assembly (e.g., packing of microscale hydrogel particles within a casing to form a porous structure).

[0083] In some embodiments, the porous cell bed comprises a porous ceramic scaffold. Any suitable method known to the skilled artisan for forming a porous ceramic scaffold may be used herein. In some embodiments, the methods include, but are not limited to, foam replication (e.g., coating a polymer foam with ceramic slurry and burn off the polymer, leaving a porous ceramic structure) and / or slip casting with porogen (e.g., add a pore-forming agent, such as starch or wax, to a ceramic slurry, then remove them post-sintering).

[0084] In some embodiments, the porous bed comprises a decellularized natural tissue. Any suitable decellularized natural tissue may be used as a porous cell scaffold for making a 3D bio-printed liver, or other organ. Further, in some embodiments, any suitable enzymatic and / or detergent-based decellularization protocol may be used to produce the decellularized natural tissue. In some embodiments, the decellularized natural tissue comprises lung, intestine, skin, muscle, pancreas, liver, stomach, kidney, brain, heart, or spleen.

[0085] In some embodiments, the porous cell bed used to make the 3D bio-printed organ comprises a microgel assembly. In some embodiments, the microgel assembly comprises a casing (e.g., a pouch, bag, or mold) loaded with a plurality of organoids encapsulated within a microgel (e.g., a organoid loaded hydrogel microparticles). In some embodiments, the methods comprise creating a casing (e.g., mesh casing) in the shape of a liver, or portion thereof, loading a plurality of microporous organoid- infused microgel matrices into the casing, and using embedded bioprinting to create a hepatic artery, a portal vein, and biliary duct within the casing. In some embodiments, the casing is a mesh casing.

[0086] In some embodiments, the methods further comprise maturing the 3D bio-printed liver in a bioreactor. In some embodiments, the methods further comprise endothelializing the hepatic artery and portal vein with ECs. In some embodiments, the methods further comprise endothelializing the biliary duct with cholangiocytes.

[0087] In some embodiments, the methods relate to fabricating a casing in the shape of a target organ or a portion thereof (e.g., a liver). In some embodiments, the methods comprise weaving the casing into a mesh-like structure. The casing may be woven into a mesh-like structure using any suitable technique known to the skilled artisan. For example, in some embodiments, the casing is woven via electrospinning, melt spinning, wet spinning, dry spinning, textile weaving or knitting, braiding or filament winding.

[0088] In some embodiments, the casing is cut into a shape of a target organ (e.g., liver) or a portion thereof (e.g., a portion of a liver). For example, in some embodiments, the casing is cut into a shape of a liver, kidney, spleen, stomach, intestine, heart, brain, pancreas, or any portion thereof. Any technique known to the skilled artisan may be used to cut the casing into the desired shape. Exemplary embodiments include, but are not limited to, laser cutting, die cutting, plotter cutting (e.g., knife or blade), waterjet cutting, punching, or CNC milling.

[0089] In some embodiments, the methods relate to creating allogenic hypoimmunogenic iPSC-derived cells (e.g., iPSC-derived liver cells) as a “universal cell” that avoids expensive patient-derived specific cell derivation and expansion (see Examples section for specific details). In some embodiments, the methods comprise scaling hiPSC-derived cell expansion and differentiation into specific cell types (e.g., liver cells). While the use of hiPSC-derived cells are contemplated herein, the invention is not limited to hiPSC-derived cells, and may be used with any suitable cell type (e.g., primary cells, cell line, stem cells, etc.).

[0090] In some embodiments, the methods relate to differentiating the cells into more or more cell type (e.g., differentiation allogenic hypoimmunogenic iPSC-derived cells into one or more target cell type). In some embodiments, the target cell types comprises: neural progenitor cells, radial glia cells, cortical neurons, astrocytes, and / or oligodendrocytes, definitive endoderm, anterior foregut endoderm, lung progenitor cells, alveolar epithelial cells, and / or airway epithelial cells, into cardiac mesoderm cells, cardiomyocytes, cardiac fibroblasts, endothelial cells, smooth muscle cells, retinal progenitor cells, retinal pigment epithelium, and / or photoreceptor cells, otic placode-like progenitor cells and / or hair precursor cells, definitive endoderm cells, gastric progenitor cells, intestinal stem cells, enterocytes, goblet cells, and / or Paneth cells, hepatic endoderm cells, hepatocytes, and / or cholangiocyte, pancreatic progenitor cells or endocrine cells, intermediate mesoderm cells, nephron progenitors cells, podocytes, and / or proximal / distal tubule cells, hemogenic endothelium, hematopoietic progenitors, macrophages, and / or dendritic cells, bone marrow-like organoids, thymic organoids, and / or immune-organoid co-cultures, surface ectoderm cells, keratinocytes, dermal fibroblasts, and / or melanocytes, gonadal progenitor cells, Sertoli and Leydig-like cells (e.g., testis), and / or granulosa-like cells (e.g., ovaries). In some embodiments, the methods comprise differentiating the iPSC-derived cells into prehepatocytes (preHeps).

[0091] In some embodiments, the methods comprise encapsulating one or more target cells (e.g., derived from IPSC cells) within a microgel. In some embodiments, the microgel is a crosslinkable microgel. The skilled artisan may use any suitable technique known in the art to form the microgels. Exemplary techniques include, but are not limited to, emulsion polymerization techniques (including inverse emulsion polymerization, Mini emulsion polymerization, microfluidic emulsion polymerization), precipitation polymerization, photopolymerization, click-chemistry crosslinking chemistry, ionic gelation, coacervation and phase separation, spray drying, stop flow lithography, photolithography, electro spraying, electrohydrodynamic jetting, and microfluidic-assisted gelation. Additional techniques include microfluidics (e.g., hydrodynamic focusing or jetting in a coaxial configuration), extrusion (e.g., electro spraying and electrospinning) or bioprinting.

[0092] In some embodiments, the methods comprise using microfluidics to encapsulate one or more target cells (e.g., derived from IPSC-cells) within a microgel. In some embodiments, the IPSC-derived cells are encapsulated within microgels comprising one or more polymers via microfluidic emulsion polymerization. In some embodiments, the one or more polymers comprise methacrylated gelatin, polyethylene glycol diacrylate, norbomene-functionalized polyethylene glycol, hyaluronic acid methacrylate, alginate, dextran methacrylate, fibrinogen methacrylate, silk fibroin methacrylate, and / or polyvinyl alcohol modified with acrylate or thiol group. In some embodiments, the one or more polymers comprise methacrylated gelatin and polyethylene glycol. In some embodiments, the polymer is polymerized using ultraviolet light (e.g., at a wavelength of 365nm).

[0093] In some embodiments, the methods comprise packing a plurality of organoid-laden microgels into a casing, e.g. such as any casing disclosed herein, and subsequently annealing at least a portion of the microgels to one another to form a plurality of inter-particle crosslinks (e.g., the microgels are crosslinked to each other). Any suitable crosslinking agent known to the skilled artisan may be used to crosslink at least a portion of the microporous organoid-infused microgel matrices within a biodegradable casing to one another to form inter-particle crosslinks. Exemplary embodiments include, but are not limited to, genipin, 1- Ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-Hydroxy succinimide)(EDC / NHS), glutaraldehyde, transglutaminase, riboflavin, thiol-Michael addition agents, photocrosslinking agents, enzymatic crosslinking agents, and tannic acid. In some embodiments, the crosslinking agent is genipin.

[0094] In some embodiments, the methods comprise bioprinting a plurality of channels within a casing comprising a plurality of organoid microgels. In some embodiments, crosslinking at least a portion of the organoid microgel is performed prior to bioprinting a plurality of channels within the casing. In other embodiments, the crosslinking at least a portion of the microporous organoids is performed after bioprinting a plurality of channels within the biodegradable casing.

[0095] The plurality of channels, in some embodiments, are intended to mimic a native vasculature of the organ to be bio-printed. For example, in the case of a bio-printed liver, the plurality of channels includes, but is not limited to, a hepatic artery, portal vein, and / or a biliary duct. Other channel structures are possible in other embodiments.

[0096] Any suitable technique known to the skilled artisan may be used to create the plurality of channels disclosed herein. For example, in some embodiments, embedded bioprinting may be used to generate larger vessels (e.g., hepatic artery and bile networks). Embedded bioprinting is a technique in which sacrificial materials and cells can be printed within a supportive matrix (e.g., MAP annealed gels containing organoids) that provides structural support during the printing process. In some embodiments, embedded bioprinters are used to generate distinct channel networks (e.g., inputs for portal vein and hepatic artery and outputs for bile duct and hepatic vein), for example, by using alginate and Pluronic bioinks (Fig. IB). Those of skill in the art will understand and appreciate, however, that the invention is not limited to only alginate and Pluronic bioinks, and any suitable bioink may be used herein. In some embodiments, the printed networks connect an input and output port of a casing to the annealed organoid micro gels. In some embodiments, the channels are endothelialized with ECs and cholangiocytes, for example, as needed to support the maturation process (Fig. 1C).

[0097] In some embodiments, a portion of the channels having an outer diameter of greater than 2 mm (OD) are printed via porous bed co-axial bio-printing. Without wishing to be bound by a particular theory, it is generally known in the art that co-axial bio-printing uses a printer head comprising two or more nozzles, arranged one inside the other and sharing a common central axis, to produces channels with core- shell structures. The channel wall is formed via extrusion of a non- sacrificial bioink through the shell nozzle, wherein the bioink is configured to rapidly anneal after printing. The hollow interior of the channel is formed via first extruding a sacrificial bioink through the core nozzle followed by removal of said sacrificial bioink (e.g., via washing).

[0098] In some embodiments, a portion of the channels having an outer diameter of between 700 microns and 2 mm (OD) are printed using porous bed sacrificial particle bioprinting. Without wishing to be bound by a particular theory, it is generally understood that porous bed sacrificial particle bioprinting may be accomplished in at least two distinct ways. The first approach uses a 3D bio-printer to print a bioink comprising a plurality of sacrificial particles dispersed within a polymerizable fluid (e.g., the polymerizable fluid is polymerized as the bioink is printed) to print a series of channels having the desired printing path within a casing comprising the organoid-infused microgels. Dissolution of the sacrificial particles (e.g., via washing) produces channels having the desired core-shell structure.

[0099] In the second approach, a polymerizable sacrificial bioink is used to print a series of overlapping sacrificial particles having the desired printing path within a casing comprising the organoid-infused microgels. The organoid- infused microgels within the casing are then crosslinked (e.g., using genipin) to create a microporous organoid-infused microgel matrix. Subsequent dissolution of the polymerizable sacrificial bioink (e.g., alginate) produces continuous channels having a pearl-necklace-like structure within the microporous organoid- infused microgel matrix.

[0100] Any suitable bioink (e.g., sacrificial and / or non- sacrificial bioink) known to the skilled artisan may be used to print any of the vascular channels disclosed herein. In some embodiments, a sacrificial bioink comprises alginate. In some embodiments, a sacrificial bioink comprises a Pluronic co-block or Pluronic tri-block copolymer. In some embodiments, a non- sacrificial bioink comprises thiolated-gelatin (GelSH) and polyethylene- 4-arm-maleimide.

[0101] In addition to the 3D bio-printed channels discussed above, in some embodiments, a 3D bio-printed organ (e.g., liver) comprises at least a portion of channels having an outer diameter of between 5 and 50 microns. In some embodiments, the channels are formed via the interstitial spaces between the plurality of microporous organoid-infused microgel matrices contained within a biodegradable casing (e.g., a mesh casing). In some embodiments, at least a portion of the channels have an outer diameter of between 0.005 mm and 50 mm. In some embodiments, at least a portion of the channels have an outer diameter of greater than or equal to 0.005 mm, greater than or equal to 10 mm, greater than or equal to 20 mm, greater than or equal to 30.005 mm or greater than or equal to 40 mm. In some embodiments, at least a portion of the channels have an outer diameter of less than or equal to 50 mm, less than or equal to 40 mm, less than or equal to 30 mm, less than or equal to 20 mm or less than or equal to 10 mm. Combinations are also possible in some embodiments. For example, in some embodiments, at least a portion of the channels have an outer diameter of greater than or equal to 0.005 mm and less than or equal to 50 mm. Combinations of other ranges are also possible (e.g., greater than or equal to 0.005 mm and less than or equal to 50. mm). Other ranges are also possible. For example, in some embodiments, at least a portion of the channels have an outer diameter of greater than 2 mm (OD). In some embodiments, at least a portion of the channels have an outer diameter of between 700 microns and 2 mm (OD).

[0102] In some embodiments, the methods further comprise endothelializing a plurality of channels (e.g., vasculature) within a 3D bio-printed organ (e.g. liver) with one or more cell types. In some embodiments, the plurality of channels is endothelialized with induced endothelial cells (iECs). In some embodiments, a plurality of channels (e.g., vasculature) within a 3D bio-printed organ (e.g. liver) are endothelialized with primary endothelial cells.

[0103] In some embodiments, the methods comprise bioprinting a liver. In some embodiments, the methods comprise printing a hepatic vasculature within the liver. In some embodiments, the hepatic vasculature comprises a hepatic artery, a portal vein, and / or a bile duct. In some embodiments, the hepatic artery and / or the portal vein comprises iECs (e.g., are endothelialized with iECs). In some embodiments, the bile duct comprises induced cholangiocytes (iChols) (e.g., are endothelialized with iChols). In some embodiments, the hepatic artery, portal vein, and the bile duct are parallel to each other.

[0104] In some embodiments, the process of bioprinting a plurality of channels comprises obtaining a computer assisted drawing (CAD) model of the vasculature of a target organ. The CAD model can be obtained from any suitable imaging modality known to the skilled artisan such as, for example, computed tomography (CT), magnetic resonance imaging (MRI), or positron emission tomography (PET). In some embodiments, the methods comprise augmenting the CAD model using a slicer software to obtain layer-by-layer coordinates of the tissue vasculature of the target organ. In some embodiments, the methods comprise overlaying the layer-by-layer coordinates with a 3D model of a target organ geometry (e.g., the geometry of the casing loaded with the organoid microgels) and using a bioprinting software to print the target vasculature within the casing using a bioprinter (e.g., a robotic arm bioprinter shown in Figure. 9).

[0105] In some embodiments, a 3D bioprinted liver, or other organ, is matured in a commercial organ perfusion bioreactor (e.g., Harvard Apparatus for mouse size livers and TransMedics for human size bio-printed livers). Such bioreactors are known in the art to generate pulsatile flow mimicking in vivo hemodynamics which are useful for maturing the tissues (Fig. ID). In some embodiments, the tissues may further mature in vivo.

[0106] In addition, certain embodiments are directed to using organs such as those described herein. For example, an organ such as a 3D bio-printed liver may be transplanted into a subject, e.g., in need thereof. The subject may be human, or a non-human animal. Examples of subjects include, but are not limited to, a mammal such as a cow, sheep, goat, horse, rabbit, pig, mouse, rat, dog, cat, a primate (e.g., a monkey, a chimpanzee, etc.), or the like. In some cases, the subject is a non-mammal such as a bird, an amphibian, or a fish. In some embodiments, the subject is genetically engineered.

[0107] The following numbered clauses are exemplary embodiments of the disclosure and are not intended to be limiting.

[0108] Clause 1. A 3D bio-printed liver, comprising: a porous cell bed; and a hepatic vasculature contained within the porous cell bed.

[0109] Clause 2. A 3D bio-printed organ, comprising: A porous cell bed in a shape of a target organ, or fragment thereof; and a vasculature contained within the porous cell bed.

[0110] Clause 3. A method for producing a 3D bio-printed liver, or portion thereof, comprising: creating a porous cell bed in a shape of liver, or portion thereof; and using embedded bioprinting to create a hepatic artery, a portal vein, and biliary duct within a mesh casing. Clause 4. A method for producing a 3D bio-printed organ, or portion thereof, comprising: creating a porous cell bed in a shape of liver, or portion thereof; and using embedded bioprinting to create an organ vasculature within the porous cell bed.

[0111] Clause 5. A 3D bio-printed liver, comprising: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a hepatic vasculature within the mesh casing.

[0112] Clause 6. The 3D bio-printed liver of clause 5, wherein the mesh casing comprises poly (glycerol sebacate).

[0113] Clause 7. The 3D bio-printed liver of clause 5 or 6, wherein the mesh casing comprises an open top. Clause 8. The 3D bio-printed liver of any one of clauses 5-7, wherein the mesh casing comprises one or more suturable ports.

[0114] Clause 9. The 3D bio-printed liver of any one of clauses 5-8, wherein the plurality of microporous organoid-infused microgel matrices comprises one or more micro-architectured polymeric (MAP) micro-annealed gels.

[0115] Clause 10. The 3D bio-printed liver of clause 9, wherein the one or more MAP microannealed gels comprise one or more hiPSC-derived liver organoids.

[0116] Clause 11. The 3D bio-printed liver of any one of clauses 5-10, wherein at least a portion of the plurality of microporous organoid-infused microgel matrices are crosslinked to one another, thus forming inter-particle crosslinks.

[0117] Clause 12. The 3D bio-printed liver of clause 11, wherein at least a portion the plurality of microporous organoid-infused microgel matrices are crosslinked using a crosslinking agent. Clause 13. The 3D bio-printed liver of clause 12, wherein the crosslinking agent is genipin. Clause 14. The 3D bio-printed liver of clause 13, wherein the genipin is at a concentration of between 0.1 % and 1 % (wt / wt).

[0118] Clause 15. The 3D bio-printed liver of any one of clauses 5-14, wherein the hepatic vasculature comprises a plurality of channels.

[0119] Clause 16. The 3D bio-printed liver of clause 15, wherein at least a portion of the channels have an outer diameter of greater than 2 mm (OD).

[0120] Clause 17. The 3D bio-printed liver of clause 15, wherein at least a portion of the channels have an outer diameter of between 700 microns and 2 mm (OD).

[0121] Clause 18. The 3D bio-printed liver of clause 16, wherein the portion of channels having an outer diameter of greater than 2 mm (OD) are printed via porous bed co-axial bio-printing. Clause 19. The 3D bio-printed liver of clause 18, wherein the porous bed co-axial bioprinting produces a channel comprising a core- shell structure, the core comprising a sacrificial bioink and the shell comprising a non- sacrificial bioink.

[0122] Clause 20. The 3D bio-printed liver of clause 19, wherein the sacrificial bioink (core) comprises alginate.

[0123] Clause 21. The 3D bio-printed liver of clause 19 or 20, wherein the non- sacrificial bioink (shell) comprises thiolated-gelatin (GelSH) and polyethylene-4-arm-maleimide.

[0124] Clause 22. The 3D bio-printed liver of clause 17, wherein the portion of channels having an outer diameter of between 700 microns and 2 mm (OD) are printed using porous bed sacrificial particle bio-printing. Clause 23. The 3D bio-printed liver of clause 20, wherein porous bed sacrificial particle bioprinting uses a second sacrificial bioink comprising alginate.

[0125] Clause 24. The 3D bio-printed liver of clause 15, wherein at least a portion of the channels have an outer diameter of between 5 and 50 microns.

[0126] Clause 25. The 3D bio-printed liver of clause 24, wherein the channels are formed via interstitial spaces between the plurality of microporous organoid-infused microgel matrices contained within the mesh casing.

[0127] Clause 26. The 3D bio-printed liver of any one of clauses 15-25, wherein the plurality of channels comprises induced endothelial cells (iECs).

[0128] Clause 27. The 3D bio-printed liver of any one of clauses 5-25, wherein the hepatic vasculature comprises a hepatic artery.

[0129] Clause 28. The 3D bio-printed liver of any one of clauses 5-27, wherein the hepatic vasculature comprises a portal vein.

[0130] Clause 29. The 3D bio-printed liver of clause 27 or 28, wherein the hepatic artery and / or the portal vein comprise iECs.

[0131] Clause 30. The 3D bio-printed liver of any one of clauses 5-28, wherein the hepatic vasculature comprises a bile duct.

[0132] Clause 31. The 3D bio-printed liver of clause 30, wherein the bile duct comprises induced cholangiocyte cells (iChols).

[0133] Clause 32. The 3D bio-printed liver of any one of clauses 27-30, wherein the hepatic artery, portal vein, and the bile duct are parallel to each other.

[0134] Clause 33. A method for producing a 3D bio-printed liver, comprising: creating a mesh casing in a shape of a liver, or portion thereof; loading a plurality of microporous organoid- infused microgel matrices into the mesh casing; and using embedded bioprinting to create a hepatic artery, a portal vein, and biliary duct within the mesh casing.

[0135] Clause 34. The method of clause 33, further using a crosslinking agent crosslink at least a portion of the microporous organoid-infused microgel matrices to one another to form interparticle crosslinks.

[0136] Clause 35. The method of clause 34, wherein the crosslinking agent is genipin.

[0137] Clause 36. The method of any one of clauses 33-35, wherein the embedded bioprinting comprises co-axial bioprinting to create the hepatic artery, portal vein, and biliary duct. Clause 37. The method of clause 36, wherein co-axial printing comprises a sacrificial bioink and a non-sacrificial bioink to create channels having a core-shell structure and outer diameters (OD) greater than 2 mm. Clause 38. The method of clause 37, wherein the sacrificial bioink (core) comprises alginate. Clause 39. The method of clause 37 or 38, wherein the non- sacrificial bioink (shell) comprises thiolated-gelatin (GelSH) and polyethylene-4-arm-maleimide.

[0138] Clause 40. The method of any one of clauses 33-39, wherein the embedded bioprinting further comprises sacrificial particle printing to create channels between 700 microns and 2 mm (OD).

[0139] Clause 41. The method of clause 40, wherein the sacrificial particle printing uses a second sacrificial bioink to create a series of overlapping sacrificial particles having a first printing path.

[0140] Clause 42. The method of clause 41, wherein the second sacrificial bioink comprises alginate. Clause 43. The method of any one of clauses 40-42, further comprising incubating the 3D bio-printed liver in an aqueous solution to remove the second sacrificial bioink to produce a continuous channel having a pearl-necklace-like structure.

[0141] Clause 44. The method of clause 43, wherein the aqueous solution comprises sodium ions (Na+).

[0142] Clause 45. The method of any one of clauses 33-44, wherein loading the plurality of microporous organoid- infused microgel matrices into the mesh casing creates a plurality of interstitial spaces between adjacent microporous organoid- infused microgel matrices.

[0143] Clause 46. The method of clause 45, further comprising endothelializing the plurality of interstitial spaces with iECs to create capillary networks.

[0144] Clause 47. The method of any one of clauses 33-46, further comprising endothelializing the hepatic artery and portal vein with iECs or primary ECs.

[0145] Clause 48. The method of any one of clauses 33-47, further comprising endothelializing the biliary duct with iChols or primary cholangiocytes.

[0146] Clause 49. The method of any one of clauses 33-48, further comprising maturing the 3D bioprinted liver in a bioreactor.

[0147] Clause 50. A 3D bio-printed organ, comprising: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a vasculature within the mesh casing.

[0148] Clause 51. The 3D bio-printed organ of clause 50, wherein the mesh casing comprises poly (glycerol sebacate).

[0149] Clause 52. The 3D bio-printed organ of clause 50 or 51, wherein the mesh casing comprises a coating. Clause 53. The 3D bio-printed organ of clause 52, wherein the coating comprises an antifouling material.

[0150] Clause 54. The 3D bio-printed organ of clause 53, wherein the antifouling material comprises hyaluronic acid methacrylate (HAMA) and hydrosulfuryl heparin (HepSH).

[0151] Clause 55. The 3D bio-printed organ of any one of clauses 50-54, wherein the mesh casing has a burst pressure of between 100 mmHg and 160 mmHg.

[0152] Clause 56. The 3D bio-printed organ of any one of clauses 50-55, wherein the organoids within the plurality of microporous organoid-infused microgel matrices are selected from the group consisting of liver organoids, lung organoids, retinal organoids, intestinal organoids, gastric organoids, kidney organoids, cardiac organoids, and tumor organoids.

[0153] Clause 57. The 3D bio-printed organ of any one of clauses 50-56, wherein the microgel matrices within the plurality of microporous organoid-infused microgel matrices comprise methacrylated gelatin, polyethylene glycol diacrylate, norbornene-functionalized polyethylene glycol, hyaluronic acid methacrylate, alginate, dextran methacrylate, fibrinogen methacrylate, silk fibroin methacrylate, and / or polyvinyl alcohol modified with acrylate or thiol group.

[0154] Clause 58. The 3D bio-printed organ of any one of clauses 50-57, wherein at least a portion of the plurality of microporous organoid-infused microgel matrices are crosslinked to one another to form inter-particle crosslinks.

[0155] Clause 59. The 3D bio-printed organ of any one of clauses 50-58, wherein the vasculature comprises vessels with outer diameters greater than 2 mm (OD).

[0156] Clause 60. The 3D bio-printed organ of any one of clauses 50-59, wherein the vasculature comprises vessels with outer diameters between 700 microns and 2 mm (OD).

[0157] Clause 61. The 3D bio-printed organ of any one of clauses 50-60, wherein the vasculature comprises vessels with outer diameters of between 5 microns and 50 microns (OD).

[0158] Clause 62. The 3D bio-printed organ of clause 61, wherein the vessels having outer diameters of between 5 microns and 50 microns (OD) are formed via interstitial spaces between adjacent microporous organoid-infused microgel matrices within the mesh casing.

[0159] Clause 63. The 3D bio-printed organ of any one of clauses 50-62, wherein the vasculature comprises a hepatic artery, a portal vein, a biliary duct and / or a capillary network.

[0160] Clause 64. The 3D bio-printed organ of any one of clauses 50-63, wherein the mesh casing comprises one or more suture-able ports.

[0161] Clause 65. The 3D bio-printed organ of clause 64, wherein the one or more suture-able ports are in fluidic communication with hepatic artery, portal vein, or biliary duct. Clause 66. The 3D bio-printed organ of clause 63, wherein the hepatic artery, portal vein, and / or capillary network comprise iECs or primary ECs.

[0162] Clause 67. The 3D bio-printed organ of clause 63, wherein the biliary duct comprises iChol cells or primary cholangiocytes.

[0163] Clause 68. A method for producing a 3D bio-printed organ, comprising: creating a mesh casing in a shape of an organ, or portion thereof; loading a plurality of microporous organoid- infused microgel matrices into the mesh casing; and using embedded bioprinting to create an organ vasculature within the mesh casing.

[0164] Clause 69. The method of clause 68, further comprising using a crosslinking agent to crosslinking at least a portion of the plurality of microporous organoid-infused microgel matrices to one another to produce a porous bed comprising a plurality of inter-particle crosslinks.

[0165] Clause 70. The method of clause 69, wherein the crosslinking agent is selected from the group consisting of genipin, l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide and N- Hydroxy succinimide) (EDC / NHS), glutaraldehyde, transglutaminase, riboflavin, thiol- Michael addition agents, photo-crosslinking agents, enzymatic crosslinking agents, and tannic acid.

[0166] Clause 71. The method of clause 70, wherein the thiol-Michael addition agents comprise PEG-maleimide or PEG- vinyl sulfone.

[0167] Clause 72. The method of any one of clauses 69-71, wherein the porous bed has a storage modulus of between 10 Pa and 106 Pa.

[0168] Clause 73. The method of any one of clauses 69-72, wherein the porous bed has a loss modulus of between 1 Pa and 104 Pa.

[0169] Clause 74. The method of any one of clauses 68-73, further comprising obtaining computed tomography (CT) images of the organ vasculature.

[0170] Clause 75. The method of clause 74, further comprising obtaining a 3D model of the mesh casing.

[0171] Clause 76. The method of clause 74 or 75, further comprising using a computer automated design software to slice the organ vasculature and 3D model of mesh casing into layers to obtain a plurality of layer-by-layer coordinates.

[0172] Clause 77. The method of clause 76, wherein the plurality of layer-by-layer coordinates are used by a robotic embedded bioprinting software to print the organ vasculature within the mesh casing. Clause 78. The method of any one of clauses 68-77, wherein at least a portion of the organ vasculature has an outer diameter of greater than 2 mm printed using co-axial bioprinting. Clause 79. The method of any one of clause 68-77, wherein at least a portion of the organ vasculature has an outer diameter of between 700 micron and 2 mm printed using sacrificial particle printing.

[0173] Clause 80. The method of any one of clauses 68-79, wherein the organ vasculature comprises channels with outer diameters of between 5 microns and 50 microns (OD), the channels formed via interstitial spaces between adjacent microporous organoid- infused microgel matrices within the mesh casing.

[0174] Clause 81. The method of any one of clauses 68-80, wherein the mesh casing comprises one or more suture-able ports in fluidic communication with at least a portion of the organ vasculature.

[0175] Clause 82. The method of clause 80, further comprising seeding ECs and / or iChol cells within at least a portion of the organ vasculature by perfusing an EC solution and / or iChol solution through at least a portion of the organ vasculature.

[0176] Clause 83. The method of any one of clauses 68-82, further comprising maturing the 3D bioprinted organ in a bioreactor.

[0177] Clause 84. A 3D bio-printed organ, comprising: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a vascular network, wherein a portion of the vascular network comprises a plurality of channels having an outer diameter (OD) greater than 2 mm.

[0178] Clause 85. A 3D bio-printed organ, comprising: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a vascular network, wherein a portion of the vascular network comprises a plurality of channels having an OD of between 700 microns and 2 mm.

[0179] Clause 86. A 3D bio-printed organ, comprising: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a vascular network, wherein a portion of the vascular network comprises a plurality of channels having an OD of between 5 microns and 50 microns.

[0180] Clause 87. A 3D bio-printed organ, comprising: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a vascular network, wherein the vascular network comprises: (i) a first plurality of channels having an outer diameter (OD) of greater than 2 mm, and (ii) a third plurality of channels having an OD of between 5 microns and 50 microns. Clause 88. A 3D bio-printed organ, comprising: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a vascular network, wherein the vascular network comprises: (i) a second plurality of channels having an outer diameter (OD) of between 700 microns and 2 mm, and (ii) a third plurality of channels having an OD of between 5 microns and 50 microns.

[0181] Clause 89. A 3D bio-printed organ, comprising: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a vascular network, wherein the vascular network comprises: (i) a first plurality of channels having an outer diameter (OD) of greater than 2 mm, and (ii) a second plurality of channels having an OD of between 700 microns and 2 mm.

[0182] Clause 90. A 3D bio-printed organ, comprising: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a vascular network, wherein the vascular network comprises: (i) a first plurality of channels having an outer diameter (OD) of greater than 2 mm, (ii) a second plurality of channels having an OD of between 700 nm and 2 mm, and (iii) a third plurality of channels having an OD of between 5 microns and 50 microns.

[0183] Clause 91. The 3D bio-printed organ of any one of clauses 84-90, wherein the 3D bio-printed organ is selected from the group consisting of liver, kidney, heart, lung, spleen, brain, skin, pancreas, and intestine.

[0184] Clause 92. A method for producing a 3D bio-printed organ, comprising: creating a mesh casing in a shape of a target organ, or portion thereof; loading a plurality of microporous organoid-infused microgel matrices into the mesh casing; and using embedded bioprinting to create a vascular network comprising a plurality of channels having an OD of greater than 2 mm.

[0185] Clause 93. A method for producing a 3D bio-printed organ, comprising: creating a mesh casing in a shape of a target organ, or portion thereof; loading a plurality of microporous organoid-infused microgel matrices into the mesh casing; and using embedded bioprinting to create a vascular network comprising a plurality of channels having an OD of between 700 microns and 2 mm.

[0186] Clause 94. A method for producing a 3D bio-printed organ, comprising: creating a mesh casing in a shape of a target organ, or portion thereof; loading a plurality of microporous organoid-infused microgel matrices into the mesh casing; and using embedded bioprinting to create a vascular network comprising a plurality of channels having an OD of between 5 microns and 50 microns. Clause 95. A method for producing a 3D bio-printed organ, comprising: creating a mesh casing in a shape of a target organ, or portion thereof; loading a plurality of microporous organoid-infused microgel matrices into the mesh casing; and using embedded bioprinting to create a vascular network, wherein the vascular network comprises: (i) a first a plurality of channels having an OD of between 5 microns and 50 microns, and (ii) a third plurality of channels having an OD of between 5 microns and 50 microns.

[0187] Clause 96. A method for producing a 3D bio-printed organ, comprising: creating a mesh casing in a shape of a target organ, or portion thereof; loading a plurality of microporous organoid-infused microgel matrices into the mesh casing; and using embedded bioprinting to create a vascular network, wherein the vascular network comprises: (i) a second plurality of channels having an outer diameter (OD) of between 700 microns and 2 mm, and (ii) a third plurality of channels having an OD of between 5 microns and 50 microns.

[0188] Clause 97. A method for producing a 3D bio-printed organ, comprising: creating a mesh casing in a shape of a target organ, or portion thereof; loading a plurality of microporous organoid-infused microgel matrices into the mesh casing; and using embedded bioprinting to create a vascular network, wherein the vascular network comprises: (i) a first plurality of channels having an outer diameter (OD) of greater than 2 mm, and (ii) a second plurality of channels having an OD of between 700 microns and 2 mm.

[0189] Clause 98. A method for producing a 3D bio-printed organ, comprising: creating a mesh casing in a shape of a target organ, or portion thereof; loading a plurality of microporous organoid-infused microgel matrices into the mesh casing; and using embedded bioprinting to create a vascular network, wherein the vascular network comprises: (i) a first plurality of channels having an outer diameter (OD) of greater than 2 mm, (ii) a second plurality of channels having an OD of between 700 nm and 2 mm, and (iii) a third plurality of channels having an OD of between 5 microns and 50 microns.

[0190] Clause 99. The method of any one of clauses 92-98, wherein the 3D bio-printed organ is selected from the group consisting of liver, kidney, heart, lung, spleen, brain, skin, pancreas, and intestine.

[0191] U.S. Provisional Patent Application Serial No. 63 / 686,458, filed August 23, 2024, entitled “3D Bioprinted Liver,” is incorporated herein by reference in its entirety.

[0192] The following examples are intended to illustrate certain embodiments of the present disclosure, but do not exemplify the full scope of the disclosure.

[0193] EXAMPLE 1 This example illustrates methods for generation and biobanking of liver- specific cells from hiPSCs, according to some embodiments. This example also demonstrates GLP manufacturing procedures of liver- specific differentiated cell types and establishment of a master biobank. In some embodiments, the methods comprise manufacturing and assembling functional bio-printed liver lobules and performing embedded bioprinting to generate the implantable liver. In some embodiments, the methods comprise perfusing the bio-printed liver, for example, using an ex vivo perfusion system to confirm its functionality, suturability, and surgical implantability. In some embodiments, the methods comprise validating the bioprinted liver in vivo, for example, by performing orthotopic humanized mouse liver transplantations and assess the safety, immunogenicity, and efficacy of said bio-printed livers under different immunosuppression regimens. The methods, according to some embodiments, further comprise establishing and validating GLP / GMP manufacturing of liverspecific cells. This may be accomplished, for example, by implanting a functional bio-printed liver in an orthotopic humanized pig liver transplantation model and evaluating its function under different immunosuppression regimens.

[0194] EXAMPLE 2

[0195] This example illustrates methods for engineering hypoimmunogenic iPSC-derived liver cells. In some embodiments, the methods use engineered hiPSCs cells, for example, hiPSCs engineered (e.g., via gene editing techniques) to knock out B2M. In some embodiments, the methods comprise validating the hypoimmunogenicity by co-culturing human T cells or NK cells with WT, B2M knockout, or B2M_ / 7 HLA-E+19-9-11 iPSCs, which demonstrated minimal T cells or NK cells mediated rejections in vitro (Fig. 2). These results were confirmed in vivo using immunodeficient NOD-scid IL2Rgnull (NSG) mice transplanted with iPSCs and challenged with human PBMCs. In some embodiments, a liver organoid or an individual cell type (e.g., within an organoid) comprises a hiPSCs. In some embodiments, hiPSCs are used to generate liver organoids or individual cell types (e.g., AFP+hepatocytes, PDGFR+stellate cells, CD31+ECs, CK19+cholangiocytes, and CD206+Kupffer cells). In some embodiments, the hiPSCs are used to generate liver organoids or individual cell types (e.g., AFP+hepatocytes, PDGFR+stellate cells, CD31+ECs, CK19+cholangiocytes, and CD206+Kupffer cells) with a yield of about IxlO9cells / day (Fig. 3). In some embodiments, the methods comprise validating one or more features of the bio-printed livers, organoids, or cells. Exemplary features include, but are not limited to, morphology, phenotype, gene expression, and function (cell secretome, nitrogen, and drug metabolism). hiPSC-derived hepatocytes were matured using defined matrix compositions (e.g. Collagen 1 / Laminin or Collagen 1 / Collagen IV) along with small molecules (e.g. cAMP) expression. In some embodiments, the methods comprise confirming the hypoimmunogenicity of the cells, for example, by assessing the cytotoxicity of allogeneic T or NK cells to these cells. However, the skilled artisan will understand that any suitable method of confirming the hypoimmunogenicity of the cells may be used. In some embodiments, the methods comprise enhancing the histocompatibility of the hiPSCs, for example, by knocking out HLA class II expression, through the deletion of HLA-DRB1, -DQB1, -DPB1, -DM, or invariant chain genes. In some embodiments, the methods comprise adding one or more suicide genes, for example, for preventing tumorigenesis in vitro and in vivo.

[0196] EXAMPLE 3

[0197] This example illustrates methods for GLP manufacturing of liver- specific differentiated cell types. The inventors have developed methods to manufacture GLP / GMP compliant iPSCs. In some embodiments, these methods comprise creating iPSC banks from multiple donors and multiple clonal iPSC lines. In some embodiments, the methods comprise characterizing the iPSC banks ensure their quality, stability, safety, identity, and genetic integrity (e.g., for inclusion in a Certificate of Analysis, CoA). In some embodiments, the methods comprise using standardized and automated platforms expand and differentiate iPSC cells (e.g., in an efficient, cost-effective, and GLP / GMP-compatible manner). In some embodiments, the methods comprise scaling up the biomanufacturing processes used to generate hiPSC-derived liver- specific cells master biobanks. In some embodiments, the methods comprise using standardized QA / QC safety assays to validate the manufacturing of hypoimmunogenic liver cells (e.g., by demonstrating liver cell-specific morphology, phenotype, gene expression, and function, etc.). In some embodiments, the methods further comprise confirming the immunogenicity and safety of the liver cells in vitro and in vivo, as described elsewhere herein).

[0198] EXAMPLE 4

[0199] This example illustrates methods for engineering a microporous liver organoid- infused microgel matrix using hypoimmunogenic iPSCs. In some embodiments, the methods comprise using a scalable microfluidic production system for generating liver microgels. In some embodiments, the microfluidic production system enables the growth of liver cells encapsulated in gels. In some embodiments, the gels are sized between 300-500 micrometers. In some embodiments, the gels are composed of ECMs that promote liver cell maturation. For example, using these microgels, liver microtissues were formed by day 9 (Fig. 4A-B). The presence of typical morphological features of liver tissues, such as bile canaliculi formation was confirmed by staining tissue samples with the appropriate fluorescent biomarkers (Fig. 4C-E). The hepatocytes remained proliferative while secreting albumin and urea for weeks in culture (Fig. 4F-H).

[0200] In some embodiments, the methods comprise producing vascularized liver organoids comprising one or more cell types (e.g., hiPSC-derived hepatocytes, stellate cells, ECs, and / or Kupffer cells). In some embodiments, the methods further comprise endothelializing one or more surfaces of the microgels, for example, to promote vascularization. In some embodiments, the methods comprise loading liver cell loaded microgels into a casing. In some embodiments, one or more vascular and / or biliary networks is bio-printed in the casing comprising the liver cell loaded microgels. In some embodiments, the methods further comprise perfusing the assembly (e.g., the liver cell loaded microgels + casing + vascular / biliary networks), for example, in a bioreactor to induce tissue maturation (Fig. 5A). In some embodiments, the methods use one or more hydrogels (e.g., collagen 1 / laminin) configured to promote formation and / or support of hepatocellular function (Fig. 5B). In some embodiments, the methods comprise confirming liver cell phenotype, gene expression, function, immunogenicity, and safety of the microporous microgel matrix in vitro and in vivo.

[0201] EXAMPLE 5

[0202] This example illustrates methods for manufacturing, assembling, and validating the fully functional, perfusable, and suturable bio-printed livers. In some embodiments, the methods comprise using a 3D embedded bioprinter to print anisotropic vascular networks within supporting hydrogel matrices (Fig. 6A-C). In some embodiments, the methods comprise using the 3D embedded bioprinter to generate one or more channel networks (e.g., comprising one or more cell types). In some embodiments, the methods further comprise using cellular self-assembly to generate the capillaries and enable their connection to the main channels. For example, the inventors have shown that printed capillary channels comprising ECs within hydrogel matrix results in formation of microvessels (Fig. 5C-E). Thus, in some embodiments, bioinks containing the appropriate cell types (e.g., hypoimmune ECs and / or cholangiocytes) are used to print the vascular and bile networks.

[0203] Any method known in the art for producing a 3D map of the liver vasculature may be used to guide the 3D printers disclosed herein (e.g., micro-CT). In some embodiments, a cast of a liver vasculature is used as the model to code the printer software to generate the channels. In some embodiments, the 3D printer has a resolution of between 100 to 500 micrometers. In some embodiments, a multi-material robotic bioprinter is used to form vascular networks within a microgel matrix. In some embodiments, one or more networks are printed in a layer-by-layer manner. In some embodiments, the shortest distance between two adjacent networks is about >2 mm.

[0204] In some embodiments, the methods comprising optimizing one or more printing parameters (e.g., to generate a functional, and perfusable liver lobule). In some embodiments, flow is used to line the channel with one or more cells (e.g., ECs and / or cholangiocytes). In some embodiments, the methods comprise generating high-density vascularized livers configured to maintain function over a prolonged period (e.g., for between 24-48 days in vitro). In some embodiments, the methods comprise using an organ perfusion system to mature a bio-printed liver (e.g., for <30 days). Once matured, allogeneic T cells or NK cells may be used, for example, to confirm functionality, and hypoimmunogenicity, according to some embodiments. In some embodiments, the methods further relate to assessing the efficacy of the bio-printed livers, e.g., by implanting the bio-printed liver in a humanized SCID mouse liver transplant model (e.g., for >3 months).

[0205] EXAMPLE 6

[0206] This example illustrates methods for GMP manufacturing of bio-printed liver tissues and bioinks. In some embodiments, the methods relate to using GMP supply chain and facilities to bio-print livers. Additionally, in some embodiments, the methods comprise utilizing qualified QA / QC safety and efficacy assays to characterize cells, and the liver tissue.

[0207] EXAMPLE 7

[0208] This example illustrates methods for implanting a functional bio-printed liver in a subject. In some embodiments, the subject is a human, pig, humanized pig, sheep, cow, etc. In some embodiments, the methods are directed toward validating the bio-printed liver in a subject. Accordingly, in some embodiments, the methods comprise implanting the bioprinted liver in a humanized pig liver transplantation model. In other embodiments, the methods comprise implanting a human- sized GMP bio-printed liver into an orthotopic immunologically humanized SCID pig model. In some embodiments, the methods comprise determining one or measures of liver function (e.g., ALT, AST, ALP, creatinine, etc.) over a prolong time period (e.g., for at least 6 months).

[0209] EXAMPLE 8

[0210] Prophetic example for the expansion ofhiPSCs. hiPSCs will be produced on a large scale using 3L Vertical- Wheel® bioreactors with a media dialysis system. hiPSCs will be placed into a 0.1 L bioreactors at a concentration of 2xl06cells with each bioreactor containing 55 mL of pre- incubated StemFlex media supplemented with 10 microM Rockl. The bioreactors will be maintained at a constant rotational speed of 60 rpm. After 24 hours, 45 mL of StemFlex media (without RockI) will be added. On day 3, 50 mL of the media will be replaced with fresh pre-incubated media by allowing the hiPSC clusters to settle by gravity and then removing the upper 50 mL of media. By day 5, the hiPSC clusters will be harvested for either experimental use or further expansion. This expansion process will be continued by transferring the hiPSC clusters into 0.5L and 3L bioreactors to increase the density of the clusters . A media dialysis system will be connected to the bioreactors to enable daily medium refreshment with base media. Growth factors will be introduced into the bioreactor compartment every 24hrs without replacing the conditioned medium, allowing macromolecule growth factors to accumulate. Waste products will be removed as the basal medium flows through the dialysis module, enriching the medium with essential nutrients such as glucose, iron, and amino acids. Once differentiated, the hiPSCs clusters will be cultured with T cells, NK cells, and alloantibodies to validate hypoimmunogenicity.

[0211] EXAMPLE 9

[0212] Prophetic example for the differentiation of hiPSC s into pre-hepatocytes and encapsulation within microgels. _hiPSCs will be expanded and then cultured in 3L bioreactors for 7 days to differentiate into pre-hepatocytes (preHeps). The cells will then be encapsulated and further differentiated into liver organoids. PreHeps will subsequently be encapsulated in spherical microgels made from GLP-materials with proven safety profiles (i.e., crosslinkable gelatin (GelMA) and multi-arm degradable polyethylene glycol (PEG)), using 3D- printed microfluidic droplet generators (Figs. 4 and 7). Different crosslinking densities and molecular weights of GelMA or PEG will be evaluated to ensure proper mechanical properties and cell encapsulation viability. Cell encapsulated microgels will be generated using microfluidic droplet generators with 100 channels that can make 6L / hr of microgels. The preHep cells encapsulated within the microgels will then be differentiated into liver organoids in the bioreactors for 14 days using optimizing differentiation protocols. Once differentiated, the liver organoids will be cultured with T cells, NK cells, and alloantibodies to validate hypoimmunogenicity.

[0213] EXAMPLE 10

[0214] Prophetic example for the differentiation ofhiPSCs into iChols. Incomplete maturation affects iChol engraftment and function. To overcome this challenge, the cell culture will be supplemented with 10 DM chenodeoxy cholic acid (CD A) to activate the FXR pathway, thereby enhancing the maturation of iChols. iChol functionality will be evaluated in a bioprinted bile duct model and their resilience to bile exposure using established in vitro assays. Once differentiated, the iChols will be cultured with T cells, NK cells, and alloantibodies to validate hypoimmunogenicity. The differentiation protocol will be refined by emulating key stages of human biliary development. After successful differentiation, iChol maturity will be evaluated by analyzing pathways, such as the FXR, and conducting transcriptomic studies from snRNAseq.

[0215] The PSXiO13 iPSC GMP line will be used to generate hiPSCs that are B2M7', CIITA" / _, HLA-E+with FailSafe switch. Components essential for EC maintenance (i.e. VEGF and bFGF) will be gradually introduced into the system, followed by nutrients for cholangiocyte differentiation (EGF). Additionally, various unified medium compositions will be subsequently tested and the resultant cell-specific phenotypes and function after maturation and expansion will be determined. All experiments will be performed under a GLP quality system in a controlled, non-classified environment under Pluristyx’s phase-appropriate standard manufacturing platform and testing. MCB and WCBs for clinical manufacturing will be generated under GMP conditions. The hiPSCs will be expanded for four passages, harvested, and cryopreserved to generate an MCB with a target size 150 vials, >1 xlO6viable cells / vial. SOPs, batch records will be maintained ensuring traceability, and electronic data management systems compliant with 21-CFR-Partl 1. To achieve a lOOx expansion, cells will be expanded in Vertical- Wheel® bioreactors (with media dialysis)

[0216] EXAMPLE 11

[0217] Prophetic example for the development of GLP protocol for hiPSCs differentiation. GLP protocols for differentiating hiPSCs into liver organoids will be developed using a 2- step approach. First, hiPSCs will be harvested, expanded, and dissociated into single cells or passaged as clumps. These cells will be seeded in a 3L PBS bioreactor for 7 days of differentiation to form preHeps. These cells will then be encapsulated in microgels to form liver organoids. After encapsulation, cells will undergo a 14-day maturation process in the bioreactor. The mature liver organoids will be harvested and undergo release testing.

[0218] EXAMPLE 12

[0219] Prophetic example for the development of GLP protocol for EC / iChols differentiation. To develop a GLP protocol for differentiating hiPSCs into ECs, electroporation will be used to deliver modRNA into cells to overexpress ETV2. CD31+cells will be separated using magnetic beads and expanded using the Corning CellCube® system for GLP scale up (LOS from Coming). This system supports high-density cultures with low shear stress and can yield up to 14 billion cells, which will be used to prepare high- density cell banks. To generate iChols we will differentiate hiPSCs in a PBS mini bioreactor. The cells will then be harvested and dissociated into iChols. EXAMPLE 13

[0220] Prophetic example for the development of mouse and pig-liver sized pouches. To ensure hemocompatibility, the pouches will be coated with the antifouling materials hyaluronic acid methacrylate (HAMA) and hydrosulfuryl heparin (HepSH). The pouches will be made with various membrane thicknesses and their mechanical integrity and ability to withstand pressures will be tested to ensure that the pouch can withstand a fluid pressure of 100-140 mmHg for small pouches used in mouse OLTs and fluid pressures of 100-160 mmHg for large pouches to be used in pig OLTs. The pouches will be subjected to incrementally increasing pressures until failure, and leakage will be assessed. Storability will be evaluated by storing pouches at various temperatures (-20°C, 4°C, and 25°C) for 1, 3, and 6 months, with checks assessing both physical integrity and maintenance of sterility. To fill the pouch with organoid-laden microgels, an incision will be made on the top. After Porous Bed printing, and the incision will be sutured and sealed with a tissue glue (Tissue Seal). To ensure physiological flow dynamics, we will establish inlet and outlet ports connected to the printed vascular network.

[0221] The pouch performance and hemocompatibility will also be assessed in vivo. The pouch will be orthotopically placed in mice, with anastomosis of the supra-hepatic inferior vena cava (IVC), portal vein (PV), intrahepatic IVC, and bile duct. Post-surgery, the pouch and blood coagulation within the pouch will be monitored for 48hrs. This will be done by euthanizing the mice, collecting the pouch, and performing immunohistochemistry staining on the 3D structure to assess any inflammatory responses or coagulation issues.

[0222] EXAMPLE 14

[0223] Prophetic example for bioprinting vasculature in mouse livers. To bioprint mouse livers, a 3D model will be used to create the bioprinting droplet printing path as well as the cast to fabricate the mouse liver pouch (Fig. 8). While normal bioprinters can make small murine livers, for large and complex structure of the human (or pig) liver, typical 3-DOF bioprinters will not easily work due to limited printing range and the inability to move the print-head to print droplets around core-shell channels. Thus, a robotic arm system will be used (Kuka LBR Med; FDA-approved) (Fig. 9) to print . This robotic arm modified bioprinter not only prints droplets to make porous mid-sized channels, but it can also print co-axial structures (i.e. core-shell) to make large vessels and the biliary network. Printer settings will be optimized by modifying droplet volume and spacing, printing speed, and needle gauge. EXAMPLE 15

[0224] Prophetic example for porous bed bioprinting. Porous Bed bioprinting requires biomaterials for: (1) organoid-laden microgels; (2) alginate bead containing sacrificial ink that generate the mid- sized channels; and (3) inks for co-axial printing of core- shell vasculature and biliary channels. Uniform spherical alginate microgels (300 micrometer) will be used to make mid-sized porous vessels via droplet printing of sacrificial channels. Alginate microgels (1-5% w / v) will be made either by spraying or a microfluidic droplet generator (Fig. 7). D is solution of the alginate beads in PBS without Ca+2will be assed to identify conditions in which they dissolve in less than 20 min. To prepare the ink for co-axial printing, which will be used for making larger blood vessels, compositions comprising thiolated gelatin (GelSH) and PEG-4arm maleimide (3-6% w / v, 1:1 ratio) will be prepared for the shell, and compositions comprising 1.5% (w / v) alginate solution will be prepared for the core. A rheometer will be used to assess the rheological properties of all components, including viscosity, shear-thinning, and yield stress. Parameters will be optimized to met the following selection criteria: induce dissolution in <20 min; (iii) >95% cell viability for cells after dissolution; (iv) Extrude microgels using >18G needles.

[0225] EXAMPLE 16

[0226] Prophetic example for crosslinking the organoid-laden microgels within the pouch. Organoid-laden microgels will be packed into a mold and visualized under confocal microscopy. It is expected that the space between the microgels will be sufficiently large for iECs infiltration (>10 micrometer). Following the optimization of packing conditions, we will test annealing of microgels by genipin, a fruit derived cytocompatibility natural crosslinker. To anneal the cell-laden GelMA microgels packed within biodegradable pouches will be exposed to 0.5% genipin. Varying genipin concentrations (0.1 - 1%) and incubation times (15 min - 2 hrs) with genipin will be tested to tune the scaffold stiffness while maintaining cell viability (>95%). Finally, the cell viability and structural integrity of the resulting scaffolds and the sacrificially printed channels will be validated. Parameters will be optimized until conditions have been identified that create stable channels (1-5 mm in diameter). This will aid in finding specific parameters required for maintaining the 3D printed structures mechanical stability during perfusion at physiological blood pressures.

[0227] EXAMPLE 17

[0228] Prophetic example for bioprinting large vessels within engineered organs. The following 3D printing scheme will be executed for large-animal and human size liver printing. Four techniques will be employed to achieve the hierarchical structure for large- animal and human-size liver printing network: (1) co-axial printing, (2) sacrificial particle printing, (3) self-assembly in the annealed microgel bed, and (4) self- assembly inside the droplet organoid.

[0229] For large vessels >2 mm in diameter. Co- axial printing (1) techniques will be used to create channels with structural walls capable of withstanding physiological pressures above 160 mm Hg. For co-axial printing of larger channels, (>2mm OD) GelSH and PEG-4- arm maleimide will be used to form the shell and alginate will be used to form the core. Key printing parameters will be optimized to include extrusion pressure (range: 10-50 kPa), printing speed (5-20 mm / s), and crosslinking conditions. The co-axial nozzle dimensions will be adjusted (OD: 2-5 mm, ID: 1-3 mm) to achieve the desired channel sizes.

[0230] Mid-sized vessels, ranging from 700 micrometer to 2 mm will be generated using sacrificial beads printed into the organoid-laden microgel bed through precision droplet-based techniques, with adjustable spacing, droplet volume, and toolpath to achieve the desired vascular topology. To generate the midsize channels (-700 micrometers-2 mm) sacrificial alginate particles will be printed into the cell-laden microgel bed to create channels. Within a bath of organoid-laden microgels, the sacrificial particles will be printed into the shape of the desired channels. The size of the sacrificial particles will be modified and the printing parameters adjusted as needed (such as spacing of droplets, volume of droplets, toolpath, nozzle diameter, and printer movement speed).

[0231] Vessels smaller than 700 micrometer will not be printed but will self-assemble in the intra- bead spaces in which endothelial cells will self- assemble to coat the surface of the microgels. The smallest capillary-scale structures (10-20 micrometers in diameter) will be formed through a self-assembly process within the organoids in the microgels.

[0232] The versatility of hierarchical vascular structures will be achieved through modifiable nozzle sizes and printing parameters, allowing iEC-coated beads to create microchannels for capillary networks after annealing (5-50 micrometers), while larger beads generate midsized channels up to 2 mm (Table 2). Finally, the sacrificial particles will be dissolved by removing Ca2+. The dissolution parameters of the sacrificial particles will be tested to ensure they do not negatively impact the liver organoids. The native liver's blood volume distribution (25-30 mL / lOOg) will be achieved through the hierarchical manufacturing approach. The system accommodates the total hepatic blood flow of 800-1200 mL / min by creating a network where 40% of the volume is held in large vessels (manufactured via co-axial printing) and 60% in the sinusoidal space (created through microgel annealing).

[0233] The bioprinting strategy for mouse liver constructs will take a similar approach to the large animal / human liver. For large vessels (200-500 micrometers in diameter), co-axial printing will be used. This technique allows creation of vessels capable of withstanding physiological pressures typical in mouse vasculature, which can range around 5-10 mmHg in the portal vein. The structural integrity of these larger vessels is required, as they must support portal blood flow rates of 1.6 to 2.3 ml / min and hepatic arterial flow rates of 0.10 to 0.35 ml / min. Mid-sized vessels (50-200 micrometers) will be generated using sacrificial droplet printing, with alginate beads within the organoid-laden microgel bed. The spacing, droplet volume, and toolpath will be adjusted to achieve a vascular topology mimicking the natural branching patterns observed in mouse liver tissue. This approach will creation of a network that can accommodate the total edge length of the mouse liver vascular network, which has been measured at approximately 861

[0234] ± 179 mm for the portal vein system and 875 ± 118 mm for the hepatic vein system in normal mice.

[0235] The smallest arterioles / venules and capillary-scale structures (10-50 micrometer and <10 micrometer), will be created using self-assembly within the porous microgel bed and inside the droplet organoid, respectively. This process will be enhanced by bioreactor perfusion fitting mouse physiological conditions, including regulation of pressure gradients which are typically less than 15 micrometer.

[0236] EXAMPLE 18

[0237] Prophetic example for creating vascular networks. To make vascular networks, a monolayer of iECs will be formed within hydrogel microchannels (both walled large channels and porous mid-size channels) as wells the surrounding microgels. The co-axial printing technique will be used to create the larger vessels (>2 mm outer diameter, which is printed using co-axial printing with the maleimide shell and alginate core. The mid-size vessels (-700 mm) will be generated using the sacrificial bead printing with alginate beads, and lastly smallest arterioles / venules and capillary-scale structures (10-50 micrometer and <10 micrometer), will be self-assembled within the porous microgel bed and inside the droplet organoid (4). Stiffer materials will be used for the larger vessels, while the mid-sized and small channels will be created using sacrificial materials in order to guide the formation of proper flow directionality. The iECs (or primary human ECs) will then be seeded within microchannels at low flow rates, and slowly perfused through the spaces between the microgels. After 1 hr, non-adherent cells will be gently washed from the microchannel, leaving the adherent cells on the gels. The process will be repeated if needed and the tissue may be rotated to ensure full confluency. Using this approach, ECs will be seeded into microchannels and flow utilized to induce shear stresses in the physiologically relevant ranges from 0.4 dyne / cm2(low) to 12 dyne / cm2(normal) to assess cell adhesion and the viability. Pulsatile rather than continuous flow will be used as a better model for physiological conditions. Shear stress will be modeled using known methods. The liver’s arterial and venous pressure balance is vital to its function, supported by dual blood supply from the hepatic artery (oxygen-rich) and portal vein (nutrient-rich). In mice, arterial pressures range from 80-100 mmHg and portal pressures from 5- 10 mmHg, while in pigs, these are 90-120 mmHg and 6-12 mmHg, closely resembling human physiology. Within hepatic sinusoids, low pressures (~3-5 mmHg) enable efficient oxygen, nutrient, and waste exchange. The liver regulates this balance via mechanisms like the hepatic arterial buffer response, ensuring metabolic function and homeostasis. Species differences, such as smaller liver size and lower blood flow in mice, highlight pigs as superior models for human liver studies.

[0238] To prevent vascular flow into the biliary network, the bile duct outlets will be closed off. The biliar network will be seeded after the vascular network is formed. It will also be formed through co-axial printing and sacrificial droplet printing to form distinct channels that separate bile flow from the vascular flow. The bile ducts will be printed with core-shell structures. iChols will be seeded in the channels (with core- shell structures to exclude vascular perfusion), similar to above. It will be ensured that inlet and outlet ports for the bile ducts do not interconnect with the vascular system.

[0239] EXAMPLE 19

[0240] Prophetic example of contemplated, perfusion systems. Exemplary contemplated perfusion systems may be microfluidic or large-scale perfusion bioreactors, such as TEB1000, and OrganOX™. The TEB1000 bioreactor is a sophisticated system for tissue maturation in ex vivo settings, enhancing scalability and flexibility. Its integrated peristaltic pumping systems control up to twenty flow channels, maintaining a complex microenvironment for liver maturation. The TEB1000 bioreactor (Ebers Inc.) has been used to perfuse vascularized tissues and to show that direct flow-induced shear stress significantly enhances vascular network formation, vessel maturation, and ECM depth distribution in 3D engineered tissues compared to static conditions. The 3D- printed mouse liver constructs will be cultured in a controlled perfusion for up to 30 days. During this period, liver- specific functions will be assessed continuously as well as evaluation of vascularization, and tissue morphology. The perfusion in the bioreactors will be used to mature the tissue to mimic the in vivo blood flow patterns critical for the formation of mature vascular networks and proper tissue function. These systems ensure that the vascularized constructs can support physiologically relevant flow rates and withstand physiological pressures (>160 mm Hg) without developing areas of stagnation or excessive shear stress.

[0241] EXAMPLE 20

[0242] Prophetic example for manufacture of liver organoids. Liver organoids will be encapsulated in microgels as described above. These organoid-laden microgels will be packed into a suturable mouse pouch as described above. The packing density will be optimized to achieve 80-90% occupancy, as determined by confocal microscopy. The integrity and overall structure will be tested through pressure leak tests and dye perfusion studies, ensuring the perfusion of the construct. For mouse, for large vessels (200-500 micrometer in diameter), we will utilize co-axial printing (1), for mid-sized vessels (50-200 micrometer) will be generated using sacrificial droplet printing (2), with alginate beads within the organoidladen microgel bed. The smallest arterioles / venules and capillary-scale structures (10-50 micrometer and < 10 micrometer), will be created by self-assembly within the porous microgel bed and inside the droplet organoid, respectively.

[0243] The inlet and outlet vessels and the biliary network will be printed with co-axial printing. After the annealing process, the liver organoid-containing pouch will be sealed. Following removal of sacrificial layers, the resulting channels will be seeded with iECs and iChols. Finally, the vascular channels will be perfused to promote self-assembly of the microvasculature within the annealed microgels. Bile duct function and continuity between the bile ducts and bile canaliculi will be assessed using fluorescent bile acid (e.g. CLF) secretion assays, transporter assays (e.g. MDR3 function using Rhodamine 123), choleresis assays using e.g. secretin, and biochemical characterization of the produced bile including quantification of bile acids, pH, bicarbonate and chloride.

[0244] EXAMPLE 21

[0245] Prophetic example for maturation of bioprinted mouse livers. Mouse bioprinted livers will be matured using a microfluidics perfusion machine. The bioprinted liver construct will be placed in a perfusion bioreactor equipped with integrated sensors. The maturation process will begin with perfusing unified media, at physiological flow rates (10-20 mL / min), while monitoring and maintaining optimal temperature (37 °C), pH (7.2-7.4), and 02 levels (5- 10%). After 3-4 weeks of unified media perfusion, the system will transition to heparinized human blood, with adjustments to flow rates and coagulation factor monitoring. Liver cell distribution and function will be determined after the 4- week maturation period. In addition, the ability to achieve stable perfusion and metabolic activity will be assessed (e.g., with physiological 02 consumption, glucose uptake, and lactate production, liver- specific protein production, including Albumin and prothrombin, hepatic enzyme functionality (i.e., ALT, AST, and CYP450), and functional biliary and a vascular network supporting sustained perfusion with heparinized blood).

[0246] EXAMPLE 22

[0247] Prophetic example for bioprinted, porcine livers. Porcine sized livers will be bioprinted using similar approaches. A perfusable vasculature and biliary network will be created using Porous Bed printing. To establish the vascular network, iECs will be perfused into the liver while keeping the bile network excluded and the common hepatic duct (i.e. bile duct outlet) closed. After seeding and maturation of the vasculature, the biliary network will be generated. The construct will undergo maturation and monitoring in a perfusion bioreactor for up to 30 days.

[0248] To assess the biocompatibility and blood coagulation of the unmodified and coated (anti-fouling) pouch, two groups will be investigated: Group 1: Non-coated pouch filled with microgels; and Group 2: Pouch filled with microgels, coated with an anti-coagulant. Normal pigs between 20-24 weeks old will be used for the study as their livers are fully grown. Postsurgery, the 3D structure's suturability and blood coagulation will be assessed after 48 hrs. Pigs will be euthanized, and the pouch collected for analysis.

[0249] To perform B-OLT experiments, RAG2 / IL2RG deficient pigs will be generated by injecting CRISPR / Cas9 RNA into early IVF-derived zygotes and performing embryo transfer into surrogate sows. Piglets will be delivered by hysterectomy and maintained in “biobubble” isolators under germ-free condition. Newborn animals will be genotyped to confirm successful knockout. For transplantation studies, 6 to 8-week-old pigs will be used, as they have relatively large livers while still being young enough to thrive in the biobubble environment.

[0250] GLP bioprinted livers will be fabricated and following maturation, the liver will be delivered to the University of Missouri via private airplane (<4 hrs) to ensure that the tissue is freshly maintained (Trinity Medical LOS). Transport on ice or via use of the OrganOx Normothermic Machine Perfusion (OrganOx LOS) will be evaluated. For OrganOx, the liver will be continuously perfused with oxygenated human blood, medications, and nutrients at normal body temperature and near-physiological pressures and flows. The machine is fully automated and can preserve the liver organ in a functional state for up to 24 hrs, allowing determination of its perfusability, viability, and function upon arrival. Immediately upon arrival, the liver will be transplanted by a physician who has extensive experience in this pig OLT. Supra hepatic vena cava, IVC, hepatic artery, and PV anastomoses will be connected to the host vessels with the engineered vessels, allowing reperfusion. The abdomen will be closed, animal extubated, and returned to the biobubble for full recovery. Animals will be monitored closely post-operatively, with vitals taken and blood drawn for liver function testing prior to implantation, during the week post- implantation, and weekly thereafter. After 3 to 6 months animals will be euthanized for full necropsy. All organs, including the B-OLT, will be analyzed by a veterinary pathologist.

[0251] EQUIVALENTSAND SCOPE

[0252] While several embodiments of the present disclosure have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present disclosure. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present disclosure is / are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the disclosure may be practiced otherwise than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the scope of the present disclosure.

[0253] In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and / or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

[0254] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms. The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0255] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0256] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”

[0257] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. When the word “about” is used herein in reference to a number, it should be understood that still another embodiment of the disclosure includes that number not modified by the presence of the word “about.”

[0258] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0259] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

What is claimed is:CLAIMS1. A 3D bio-printed liver, comprising: a porous cell bed; and a hepatic vasculature contained within the porous cell bed.

2. A 3D bio-printed organ, comprising:A porous cell bed in a shape of a target organ, or fragment thereof; and a vasculature contained within the porous cell bed.

3. A method for producing a 3D bio-printed liver, or portion thereof, comprising: creating a porous cell bed in a shape of liver, or portion thereof; and using embedded bioprinting to create a hepatic artery, a portal vein, and biliary duct within a mesh casing.

4. A method for producing a 3D bio-printed organ, or portion thereof, comprising: creating a porous cell bed in a shape of liver, or portion thereof; and using embedded bioprinting to create an organ vasculature within the porous cell bed.

5. A 3D bio-printed liver, comprising: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a hepatic vasculature within the mesh casing.

6. The 3D bio-printed liver of claim 5, wherein the mesh casing comprises poly (glycerol sebacate).

7. The 3D bio-printed liver of claim 5 or 6, wherein the mesh casing comprises an open top.

8. The 3D bio-printed liver of any one of claims 5-7, wherein the mesh casing comprises one or more suturable ports.

9. The 3D bio-printed liver of any one of claims 5-8, wherein the plurality of microporous organoid-infused microgel matrices comprises one or more micro- architectured polymeric (MAP) micro-annealed gels.

10. The 3D bio-printed liver of claim 9, wherein the one or more MAP micro-annealed gels comprise one or more hiPSC-derived liver organoids.

11. The 3D bio-printed liver of any one of claims 5-10, wherein at least a portion of the plurality of microporous organoid-infused microgel matrices are crosslinked to one another, thus forming inter-particle crosslinks.

12. The 3D bio-printed liver of claim 11, wherein at least a portion the plurality of microporous organoid-infused microgel matrices are crosslinked using a crosslinking agent.

13. The 3D bio-printed liver of claim 12, wherein the crosslinking agent is genipin.

14. The 3D bio-printed liver of claim 13, wherein the genipin is at a concentration of between 0.1 % and 1 % (wt / wt).

15. The 3D bio-printed liver of any one of claims 5-14, wherein the hepatic vasculature comprises a plurality of channels.

16. The 3D bio-printed liver of claim 15, wherein at least a portion of the channels have an outer diameter of greater than 2 mm (OD).

17. The 3D bio-printed liver of claim 15, wherein at least a portion of the channels have an outer diameter of between 700 microns and 2 mm (OD).

18. The 3D bio-printed liver of claim 16, wherein the portion of channels having an outer diameter of greater than 2 mm (OD) are printed via porous bed co-axial bio-printing.

19. The 3D bio-printed liver of claim 18, wherein the porous bed co-axial bio-printing produces a channel comprising a core-shell structure, the core comprising a sacrificial bioink and the shell comprising a non- sacrificial bioink.

20. The 3D bio-printed liver of claim 19, wherein the sacrificial bioink (core) comprises alginate.

21. The 3D bio-printed liver of claim 19 or 20, wherein the non- sacrificial bioink (shell) comprises thiolated-gelatin (GelSH) and polyethylene-4-arm-maleimide.

22. The 3D bio-printed liver of claim 17, wherein the portion of channels having an outer diameter of between 700 microns and 2 mm (OD) are printed using porous bed sacrificial particle bio-printing.

23. The 3D bio-printed liver of claim 20, wherein porous bed sacrificial particle bioprinting uses a second sacrificial bioink comprising alginate.

24. The 3D bio-printed liver of claim 15, wherein at least a portion of the channels have an outer diameter of between 5 and 50 microns.

25. The 3D bio-printed liver of claim 24, wherein the channels are formed via interstitial spaces between the plurality of microporous organoid-infused microgel matrices contained within the mesh casing.

26. The 3D bio-printed liver of any one of claims 15-25, wherein the plurality of channels comprises induced endothelial cells (iECs).

27. The 3D bio-printed liver of any one of claims 5-25, wherein the hepatic vasculature comprises a hepatic artery.

28. The 3D bio-printed liver of any one of claims 5-27, wherein the hepatic vasculature comprises a portal vein.

29. The 3D bio-printed liver of claim 27 or 28, wherein the hepatic artery and / or the portal vein comprise iECs.

30. The 3D bio-printed liver of any one of claims 5-28, wherein the hepatic vasculature comprises a bile duct.

31. The 3D bio-printed liver of claim 30, wherein the bile duct comprises induced cholangiocyte cells (iChols).

32. The 3D bio-printed liver of any one of claims 27-30, wherein the hepatic artery, portal vein, and the bile duct are parallel to each other.

33. A method for producing a 3D bio-printed liver, comprising: creating a mesh casing in a shape of a liver, or portion thereof; loading a plurality of microporous organoid-infused microgel matrices into the mesh casing; and using embedded bioprinting to create a hepatic artery, a portal vein, and biliary duct within the mesh casing.

34. The method of claim 33, further using a crosslinking agent crosslink at least a portion of the microporous organoid-infused microgel matrices to one another to form interparticle crosslinks.

35. The method of claim 34, wherein the crosslinking agent is genipin.

36. The method of any one of claims 33-35, wherein the embedded bioprinting comprises co-axial bioprinting to create the hepatic artery, portal vein, and biliary duct.

37. The method of claim 36, wherein co-axial printing comprises a sacrificial bioink and a non- sacrificial bioink to create channels having a core-shell structure and outer diameters (OD) greater than 2 mm.

38. The method of claim 37, wherein the sacrificial bioink (core) comprises alginate.

39. The method of claim 37 or 38, wherein the non- sacrificial bioink (shell) comprises thiolated-gelatin (GelSH) and polyethylene-4-arm-maleimide.

40. The method of any one of claims 33-39, wherein the embedded bioprinting further comprises sacrificial particle printing to create channels between 700 microns and 2 mm (OD).

41. The method of claim 40, wherein the sacrificial particle printing uses a second sacrificial bioink to create a series of overlapping sacrificial particles having a first printing path.

42. The method of claim 41, wherein the second sacrificial bioink comprises alginate.

43. The method of any one of claims 40-42, further comprising incubating the 3D bioprinted liver in an aqueous solution to remove the second sacrificial bioink to produce a continuous channel having a pearl-necklace-like structure.

44. The method of claim 43, wherein the aqueous solution comprises sodium ions (Na+).

45. The method of any one of claims 33-44, wherein loading the plurality of microporous organoid-infused microgel matrices into the mesh casing creates a plurality of interstitial spaces between adjacent microporous organoid- infused microgel matrices.

46. The method of claim 45, further comprising endothelializing the plurality of interstitial spaces with iECs to create capillary networks.

47. The method of any one of claims 33-46, further comprising endothelializing the hepatic artery and portal vein with iECs or primary ECs.

48. The method of any one of claims 33-47, further comprising endothelializing the biliary duct with iChols or primary cholangiocytes.

49. The method of any one of claims 33-48, further comprising maturing the 3D bioprinted liver in a bioreactor.

50. A 3D bio-printed organ, comprising: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a vasculature within the mesh casing.

51. The 3D bio-printed organ of claim 50, wherein the mesh casing comprises poly (glycerol sebacate).

52. The 3D bio-printed organ of claim 50 or 51, wherein the mesh casing comprises a coating.

53. The 3D bio-printed organ of claim 52, wherein the coating comprises an antifouling material.

54. The 3D bio-printed organ of claim 53, wherein the antifouling material comprises hyaluronic acid methacrylate (HAMA) and hydrosulfuryl heparin (HepSH).

55. The 3D bio-printed organ of any one of claims 50-54, wherein the mesh casing has a burst pressure of between 100 mmHg and 160 mmHg.

56. The 3D bio-printed organ of any one of claims 50-55, wherein the organoids within the plurality of microporous organoid-infused microgel matrices are selected from the group consisting of liver organoids, lung organoids, retinal organoids, intestinal organoids, gastric organoids, kidney organoids, cardiac organoids, and tumor organoids.

57. The 3D bio-printed organ of any one of claims 50-56, wherein the microgel matrices within the plurality of microporous organoid-infused microgel matrices comprise methacrylated gelatin, polyethylene glycol diacrylate, norbornene-functionalized polyethylene glycol, hyaluronic acid methacrylate, alginate, dextran methacrylate, fibrinogen methacrylate, silk fibroin methacrylate, and / or polyvinyl alcohol modified with acrylate or thiol group.

58. The 3D bio-printed organ of any one of claims 50-57, wherein at least a portion of the plurality of microporous organoid-infused microgel matrices are crosslinked to one another to form inter-particle crosslinks.

59. The 3D bio-printed organ of any one of claims 50-58, wherein the vasculature comprises vessels with outer diameters greater than 2 mm (OD).

60. The 3D bio-printed organ of any one of claims 50-59, wherein the vasculature comprises vessels with outer diameters between 700 microns and 2 mm (OD).

61. The 3D bio-printed organ of any one of claims 50-60, wherein the vasculature comprises vessels with outer diameters of between 5 microns and 50 microns (OD).

62. The 3D bio-printed organ of claim 61, wherein the vessels having outer diameters of between 5 microns and 50 microns (OD) are formed via interstitial spaces between adjacent microporous organoid-infused microgel matrices within the mesh casing.

63. The 3D bio-printed organ of any one of claims 50-62, wherein the vasculature comprises a hepatic artery, a portal vein, a biliary duct and / or a capillary network.

64. The 3D bio-printed organ of any one of claims 50-63, wherein the mesh casing comprises one or more suture-able ports.

65. The 3D bio-printed organ of claim 64, wherein the one or more suture-able ports are in fluidic communication with hepatic artery, portal vein, or biliary duct.

66. The 3D bio-printed organ of claim 63, wherein the hepatic artery, portal vein, and / or capillary network comprise iECs or primary ECs.

67. The 3D bio-printed organ of claim 63, wherein the biliary duct comprises iChol cells or primary cholangiocytes.

68. A method for producing a 3D bio-printed organ, comprising: creating a mesh casing in a shape of an organ, or portion thereof; loading a plurality of microporous organoid-infused microgel matrices into the mesh casing; and using embedded bioprinting to create an organ vasculature within the mesh casing.

69. The method of claim 68, further comprising using a crosslinking agent to crosslinking at least a portion of the plurality of microporous organoid-infused microgel matrices to one another to produce a porous bed comprising a plurality of inter-particle crosslinks.

70. The method of claim 69, wherein the crosslinking agent is selected from the group consisting of genipin, l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide and N- Hydroxy succinimide) (EDC / NHS), glutaraldehyde, transglutaminase, riboflavin, thiol-Michael addition agents, photo-crosslinking agents, enzymatic crosslinking agents, and tannic acid.

71. The method of claim 70, wherein the thiol-Michael addition agents comprise PEG- maleimide or PEG- vinyl sulfone.

72. The method of any one of claims 69-71, wherein the porous bed has a storage modulus of between 10 Pa and 106Pa.

73. The method of any one of claims 69-72, wherein the porous bed has a loss modulus of between 1 Pa and 104Pa.

74. The method of any one of claims 68-73, further comprising obtaining computed tomography (CT) images of the organ vasculature.

75. The method of claim 74, further comprising obtaining a 3D model of the mesh casing.

76. The method of claim 74 or 75, further comprising using a computer automated design software to slice the organ vasculature and 3D model of mesh casing into layers to obtain a plurality of layer-by-layer coordinates.

77. The method of claim 76, wherein the plurality of layer-by-layer coordinates are used by a robotic embedded bioprinting software to print the organ vasculature within the mesh casing.

78. The method of any one of claims 68-77, wherein at least a portion of the organ vasculature has an outer diameter of greater than 2 mm printed using co-axial bioprinting.

79. The method of any one of claim 68-77, wherein at least a portion of the organ vasculature has an outer diameter of between 700 micron and 2 mm printed using sacrificial particle printing.

80. The method of any one of claims 68-79, wherein the organ vasculature comprises channels with outer diameters of between 5 microns and 50 microns (OD), the channels formed via interstitial spaces between adjacent microporous organoid- infused microgel matrices within the mesh casing.

81. The method of any one of claims 68-80, wherein the mesh casing comprises one or more suture-able ports in fluidic communication with at least a portion of the organ vasculature.

82. The method of claim 80, further comprising seeding ECs and / or iChol cells within at least a portion of the organ vasculature by perfusing an EC solution and / or iChol solution through at least a portion of the organ vasculature.

83. The method of any one of claims 68-82, further comprising maturing the 3D bioprinted organ in a bioreactor.

84. A 3D bio-printed organ, comprising: a mesh casing;a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a vascular network, wherein a portion of the vascular network comprises a plurality of channels having an outer diameter (OD) greater than 2 mm.

85. A 3D bio-printed organ, comprising: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a vascular network, wherein a portion of the vascular network comprises a plurality of channels having an OD of between 700 microns and 2 mm.

86. A 3D bio-printed organ, comprising: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a vascular network, wherein a portion of the vascular network comprises a plurality of channels having an OD of between 5 microns and 50 microns.

87. A 3D bio-printed organ, comprising: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a vascular network, wherein the vascular network comprises:(i) a first plurality of channels having an outer diameter (OD) of greater than 2 mm, and(ii) a third plurality of channels having an OD of between 5 microns and 50 microns.

88. A 3D bio-printed organ, comprising: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a vascular network, wherein the vascular network comprises:(i) a second plurality of channels having an outer diameter (OD) of between 700 microns and 2 mm, and(ii) a third plurality of channels having an OD of between 5 microns and 50 microns.

89. A 3D bio-printed organ, comprising: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a vascular network, wherein the vascular network comprises:(i) a first plurality of channels having an outer diameter (OD) of greater than 2 mm, and(ii) a second plurality of channels having an OD of between 700 microns and 2 mm.

90. A 3D bio-printed organ, comprising: a mesh casing; a plurality of microporous organoid-infused microgel matrices contained within the mesh casing; and a vascular network, wherein the vascular network comprises:(i) a first plurality of channels having an outer diameter (OD) of greater than 2 mm,(ii) a second plurality of channels having an OD of between 700 nm and 2 mm, and(iii) a third plurality of channels having an OD of between 5 microns and 50 microns.

91. The 3D bio-printed organ of any one of claims 84-90, wherein the 3D bio-printed organ is selected from the group consisting of liver, kidney, heart, lung, spleen, brain, skin, pancreas, and intestine.

92. A method for producing a 3D bio-printed organ, comprising: creating a mesh casing in a shape of a target organ, or portion thereof; loading a plurality of microporous organoid-infused microgel matrices into themesh casing; and using embedded bioprinting to create a vascular network comprising a plurality of channels having an OD of greater than 2 mm.

93. A method for producing a 3D bio-printed organ, comprising: creating a mesh casing in a shape of a target organ, or portion thereof; loading a plurality of microporous organoid-infused microgel matrices into the mesh casing; and using embedded bioprinting to create a vascular network comprising a plurality of channels having an OD of between 700 microns and 2 mm.

94. A method for producing a 3D bio-printed organ, comprising: creating a mesh casing in a shape of a target organ, or portion thereof; loading a plurality of microporous organoid-infused microgel matrices into the mesh casing; and using embedded bioprinting to create a vascular network comprising a plurality of channels having an OD of between 5 microns and 50 microns.

95. A method for producing a 3D bio-printed organ, comprising: creating a mesh casing in a shape of a target organ, or portion thereof; loading a plurality of microporous organoid-infused microgel matrices into the mesh casing; and using embedded bioprinting to create a vascular network, wherein the vascular network comprises:(i) a first a plurality of channels having an OD of between 5 microns and 50 microns, and(ii) a third plurality of channels having an OD of between 5 microns and 50 microns.

96. A method for producing a 3D bio-printed organ, comprising: creating a mesh casing in a shape of a target organ, or portion thereof; loading a plurality of microporous organoid-infused microgel matrices into the mesh casing; andusing embedded bioprinting to create a vascular network, wherein the vascular network comprises:(i) a second plurality of channels having an outer diameter (OD) of between 700 microns and 2 mm, and(ii) a third plurality of channels having an OD of between 5 microns and 50 microns.

97. A method for producing a 3D bio-printed organ, comprising: creating a mesh casing in a shape of a target organ, or portion thereof; loading a plurality of microporous organoid-infused microgel matrices into the mesh casing; and using embedded bioprinting to create a vascular network, wherein the vascular network comprises:(i) a first plurality of channels having an outer diameter (OD) of greater than 2 mm, and(ii) a second plurality of channels having an OD of between 700 microns and 2 mm.

98. A method for producing a 3D bio-printed organ, comprising: creating a mesh casing in a shape of a target organ, or portion thereof; loading a plurality of microporous organoid-infused microgel matrices into the mesh casing; and using embedded bioprinting to create a vascular network, wherein the vascular network comprises:(i) a first plurality of channels having an outer diameter (OD) of greater than 2 mm,(ii) a second plurality of channels having an OD of between 700 nm and 2 mm, and(iii) a third plurality of channels having an OD of between 5 microns and 50 microns.

99. The method of any one of claims 92-98, wherein the 3D bio-printed organ is selected from the group consisting of liver, kidney, heart, lung, spleen, brain, skin, pancreas, and intestine.

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