WO2019237061A1

WO2019237061A1 - A 3d bioprinted perfusable platform for high-throughput disease modeling and drug screening

Info

Publication number: WO2019237061A1
Application number: PCT/US2019/036147
Authority: WO
Inventors: Martin Lyubomirov TOMOV; Vahid Serpooshan; Robert SCHNEPP; Wilbur Lam
Original assignee: Emory University; Children's Healthcare Of Atlanta, Inc.
Priority date: 2018-06-08
Filing date: 2019-06-07
Publication date: 2019-12-12

Abstract

Methods and systems are disclosed for a bioprinted, three-dimensional vascularized tissue device. The three-dimensional (3D) vascularized tissue device may include a perfusion chamber, a bioprinted, 3D vascular network comprised of sacrificial bioink, arranged within the perfusion chamber, and a hydrogel cast within the perfusion chamber, around the 3D vascular network. The sacrificial bioink may be removed, leaving behind a 3D structure of hollow vascular channels inside the hydrogel.

Description

A 3D BIOPRINTED PERFUSABLE PLATFORM FOR HIGH-THROUGHPUT DISEASE MODELING AND DRUG SCREENING

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No. 62/682,363, filed June 8, 2018. The provisional patent application is incorporated by reference in its entirety.

FIELD

This disclosure relates to the field of artificial organs and tissues, specifically to the use of three-dimensional (3D) bioprinting to produce a 3D perfusable, vascularized tissue device for various applications.

PARTIES TO JOINT RESEARCH AGREEMENT

Emory University and Children’s Healthcare of Atlanta are parties to a joint research agreement.

BACKGROUND

Despite advances in disease research and treatments, there remain many diseases for which the mechanisms of disease are not well known and/or treatments are not well developed or effective enough. For example, disease modeling and drug development may be ongoing for various cancers, various cardiovascular diseases, and other organ (e.g., liver) diseases. In some situations, tissue replacement may be needed to treat certain diseases.

As one example, cancer is a leading cause of death worldwide and, more specifically, pediatric tumors are the most common cause of death from disease in children in the U.S..

Approximately 10% of all new cases of invasive cancers diagnosed annually in the U.S. involve children. However, the ability of pediatric clinical trials to identify causes of specific childhood cancers and efficiently evaluate anticancer drugs is limited due to substantial heterogeneity of pediatric tumors, low incidence, and the complex microenvironment during development. Thus, there is a need to produce 3D vascularized tissue models for studying various diseases, drug screening, and in vivo tissue repair. SUMMARY

In some embodiments, a three-dimensional (3D) vascularized tissue device comprises: a perfusion chamber, a bioprinted, 3D vascular network comprised of sacrificial bioink, arranged within the perfusion chamber, and a hydrogel cast within the perfusion chamber, around the 3D vascular network.

In other embodiments, a method for manufacturing a three-dimensional (3D)

vascularized tissue device comprises: bioprinting a 3D vascular network, using a bioink material including a sacrificial bioink, within a perfusion chamber of the device, casting a hydrogel within and around the bioprinted 3D vascular network, and removing the sacrificial bioink, leaving behind a 3D structure of hollow vascular channels inside the hydrogel.

In still other embodiments, a three-dimensional (3D) vascularized tissue device comprises: a 3D network of hollow vascular channels formed inside a cast and crosslinked 3D hydrogel via degrading sacrificial bioink of a bioprinted 3D vascular network, where the hydrogel encapsulates the 3D network of hollow vascular channels and is interspersed in spaces formed between adjacent channels of the 3D network of hollow vascular channels.

The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of an embodiment of a three-dimensional (3D) bioprinting process for generating functional tissues.

FIG. 2 shows a high-level schematic of an embodiment of a process for generating a 3D vascularized tissue device, comprised of a bioprinted 3D vascular network and cast hydrogel, and perfusing the generated device using a bioreactor system.

FIG. 3 shows a flow chart of a method for manufacturing a 3D vascularized tissue device.

FIG. 4 shows an embodiment of a perfusion chamber for a perfusable, 3D vascularized tissue device.

FIG. 5 shows an example of a layer of hydrogel cast into a bottom of the perfusion chamber of FIG. 4, during manufacturing of a 3D vascularized tissue device. FIG. 6 shows an example of a 3D vascular network printed into the perfusion chamber of FIG. 4., during manufacturing of the 3D vascularized tissue device.

FIG. 7 shows an example of inserting tubing into an inlet channel and outlet channel of the perfusion chamber of FIG. 4, during manufacturing of the 3D vascularized tissue device.

FIG. 8 shows an example of casting a hydrogel within the perfusion chamber of FIG. 4, within and around an entirety of the 3D vascular network, and then crosslinking the cast hydrogel with a light source, during manufacturing of the 3D vascularized tissue device.

FIG. 9 shows the 3D vascularized tissue device, after removing the tubing from the inlet channel and outlet channel of the perfusion chamber, during manufacturing of the 3D

vascularized tissue device.

FIG. 10 shows an example process for removing sacrificial ink of the 3D vascular network, during manufacturing of the 3D vascularized tissue device.

FIG. 11 shows a final 3D vascularized tissue device, connected to a perfusion bioreactor system, following manufacturing of the 3D vascularized tissue device.

FIG. 12 shows a vascularized tissue construct including a 3D network of hollow vascular channels formed within a tissue structure, removed from the perfusion chamber after

manufacturing the 3D vascularized tissue device.

FIGS. 13A-13G show images of a 3D vascularized tissue device having a crosshatch pattern for the 3D vascular network.

FIGS. 14A-14G show different embodiments of perfusion chambers for use in a 3D vascularized tissue device having a solid organ tissue construct, for perfusion in a bioreactor system.

FIGS. 15A-15C show an embodiment of a perfusion chamber for use with a

cardiovascular or other vessel tissue construct for various flow studies.

FIGS. 16A-16D show an embodiment of a flow control valve for adjusting a flow rate of perfusion fluid through a 3D vascularized tissue device.

FIGS. 17A-17E show an example of the viability of bioprinting 3D vascular networks with a sacrificial bioink containing endothelial cells.

FIG. 18 shows a high-level schematic of an embodiment of a process for generating a 3D vascularized tissue device to study Hypoplastic left heart syndrome (HLHS).

FIGS. 19A-19B show example cross-sections of a bioprinted vascularized tissue environment of a 3D vascularized tissue device created by the process of FIG. 18, for studying HLHS. FIG. 20 shows a high-level schematic of an embodiment of a process for generating a cardiac patch for implantation in the body of a patient.

FIG. 21 shows a graph of cell viability and proliferation of endocardial cells (ECs) and cardiomyocytes (CMs) within a printed cardiac tissue patch.

FIG. 22 shows graphs demonstrating the formation of functional CMs within a cardiac tissue patch.

FIGS. 23A-23F show example devices and metabolic activity for 3D vascularized tissue devices generated with liver tissue.

DETAILED DESCRIPTION

Bioprinting technology can be used to build complex three-dimensional (3D) structures involving one or multiple cell types. It uses two disclosed features: (1) cells seeded in the form of large droplets as“bioink” that may contain several hundred to many thousands of cells mixed with degradable biomaterials, and (2) cells seeded using needles that can move in a spatially and temporally controlled user-defined manner with precision. The three-dimensional architecture in bioprinted tissue allows all the different cell types to mature simultaneously, thus increasing the likelihood of their working together like a native tissue.

However, bioprinted tissues may be limited to printable materials, thereby excluding additional types of biomaterials for forming tissue constructs. Further, bioprinting cellular material may apply high shear levels to the cells, which may result in cell damage or stress. Further, bioprinting an entire, large tissue structure for clinical applications may be time- consuming.

Disclosed herein is a 3D, vascularized tissue device (e.g., construct) that is perfusable with a fluid including culture media, blood, drugs, and/or the like. In one embodiment, the 3D vascularized tissue device includes a perfusion chamber and a chamber inlet and outlet (e.g., for introducing the fluid into and out of the perfusion chamber during perfusion), a bioprinted, 3D vascular network comprised of sacrificial bioink, arranged within the perfusion chamber; and a hydrogel cast within the perfusion chamber, around the 3D vascular network. After formation and curing (e.g., crosslinking) of the cast hydrogel, the bioink may be removed, leaving behind a 3D network of hollow channels, forming a perfusable, vascular network within the hydrogel. Thus, in another embodiment, the 3D vascularized tissue device includes a perfusion chamber and a chamber inlet and outlet, a hydrogel cast within the perfusion chamber, and a 3D hollow vascular network formed inside the cast hydrogel via removing (e.g., degrading) sacrificial bioink bioprinted within the perfusion chamber (prior to casting the hydrogel within the perfusion chamber). For example, the 3D vascularized tissue device may be generated by bioprinting a 3D network of vascular channels using a bioink material including a sacrificial bioink within a perfusion chamber of the device; casting a hydrogel within and around the 3D network of vascular channels; and degrading the sacrificial bioink, leaving behind a 3D hollow vascular structure inside the hydrogel. In some embodiments, additional cells may be seeded into individual channels of the 3D hollow vascular structure to form cells (such as endothelium) lining the hollow channels of the vascular structure. In other embodiments, additionally or alternatively, cells may be added to the hydrogel prior to casting the hydrogel inside the perfusion chamber and/or added to the sacrificial bioink prior to bioprinting the vascular channels. Following generation of the 3D vascularized tissue device, the device may be connected to a perfusion bioreactor system and various media may be perfused through the engineering vascular tissue of the 3D vascularized tissue device.

Forming a 3D vascularized tissue device with a combination of a 3D vascular network formed by bioprinting with a sacrificial bioink that is degradable and a cast hydrogel allows for the device to be produced more rapidly with a desired vascular structure. Further, a greater variety of biomaterials for forming the tissue structure may be available, while applying lower shear stresses to the cells during formation of the device, due to casting the hydrogel within the perfusion chamber, around and within the bioprinted vascular network.

As described further herein, the 3D vascularized tissue device may be used in a variety of disease modeling and drug screening applications such as in cancer modeling and screening of chemotherapy drugs, cardiovascular modeling and drug screening, and liver tissue modeling and drug screening. The 3D vascularized tissue device may also be used to generate vascularized tissue for tissue repair, such as vascularized tissue patches for cardiac repair.

Terms

Unless otherwise noted, technical terms are used according to conventional usage. In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

Bioink: A liquid, semi-solid, or solid composition for use in bioprinting. In some embodiments, bioink comprises cells, cell solutions, cell aggregates, cell -comprising gels, multicellular bodies, or tissues. In some embodiments, the bioink can be a solid or semi-solid. In some embodiments, the bioink additionally comprises non-cellular materials that provide specific biomechanical properties that enable bioprinting. In some embodiments, the bioink comprises an extrusion compound. In some cases, the extrusion compound may be in the form of a sacrificial bioink that can be removed (e.g., degraded) after the bioprinting process. In other embodiments, at least some portion of the extrusion compound remains with the cells post- printing and is not removed.

Bioprinting: Precise deposition of cells (e.g., bioink, cell solutions, cell-containing gels, cell suspensions, cell pastes, cell concentrations, multicellular aggregates, multicellular bodies, etc.) using a methodology that is compatible with an automated or semi -automated, computer- aided, three-dimensional printing device (e.g., a bioprinter). Bioprinting encompasses methods compatible with printing living cells such as an extrusion in continuous and/or discontinuous fashion. Extrusion in this context means forcing a semi-solid or solid bioink through an orifice, wherein the bioink retains its shape to a degree and for a time period after being forced through the orifice. Bioprinting also encompasses aerosol spray methods wherein cells are applied by ejecting a substantially low viscosity liquid in a mist, spray, or droplets onto a surface.

Bioprinters can be used to produce three-dimensional engineered tissue, for example by printing cells in multiple layers, layer-by-layer, on a substrate, printing cells on one or both surfaces of a substrate sheet, and/or printing multiple layers on one or both opposite surfaces of substrate sheets.

Cell: A structural and functional unit of an organism that can replicate independently, is enclosed by a membrane, and contains biomolecules and genetic material. Cells used herein may be naturally-occurring cells or artificially modified cells (e.g., fusion cells, genetically modified cells, etc.).

Hydrogel: A solid, jelly-like material having a controlled cross-linked structure exhibiting no flow when in the steady state. A hydrogel can be a water-swellable polymeric matrix that can absorb a substantial amount of water to form an elastic gel, wherein "matrices" are three-dimensional networks of macromolecules held together by covalent or noncovalent crosslinks. Upon placement in an aqueous environment, dry hydrogels swell to the extent allowed by the degree of cross-linking. A hydrogel may be comprised of gelatin methacrylate (gelMA), collagen, fibrin, and/or the like. A hydrogel may additionally contain or be mixed with a variety of cells, small molecules (e.g., growth factors), nanoparticles, imaging contrast agents, and/or the like.

Three-dimensional (3D) Vascular Network: A three-dimensional (3D) arrangement of multiple, solid (e.g., filled with sacrificial bioink) channels arranged in a desired or predetermined architecture. The predetermined architecture may include a desired shape, geometry, and/or 3D arrangement of the multiple channels. The 3D Vascular network may include a series of layers of printed channels that are spaced apart in a lateral direction and overlapping in a vertical direction, the lateral direction perpendicular to the vertical direction. The multiple channels of the 3D vascular network may be formed from a sacrificial bioink, and thus, may be filled with the sacrificial bioink. Upon removal of the sacrificial bioink, the remaining structure may be a 3D network of hollow vascular channels that may be vascularized (e.g., via the seeding or printing of endothelial cells into the channels) to create a 3D

vascularized tissue construct having vasculature.

Turning now to the figures, FIG. 1 shows a schematic 100 of an embodiment of a 3D bioprinting process for generating functional tissues. Specifically, schematic 100 outlines a traditional 3D bioprinting process for generating an engineered, functional tissue for use in vivo (e.g., in a body of a patient). The embodiment of the 3D bioprinting process begins by performing medical imaging (e.g., MRI, CT, ultrasound, or the like) on a subject (e.g., patient) at 102. At 104, the obtained medical image(s) 106 of the target anatomy of the subject undergo digital model processing to obtain a digital model (e.g., 3D computer model) 108 of the target anatomy (e.g., tissue, such as an organ or portion of an organ) of the subject. The digital model 108 may then be segmented into sections to create a segmented 3D model 110 which may be used as an input file to a bioprinting device (to enable printing, layer-by-layer, of the tissue/organ, as described further below). The process then proceeds to 112 to prepare the bioink and bioprint the tissue using the prepared bioink. For example, at 114, cells may be obtained from a cell source (such as the subject or another patient) and then combined with one or more biomaterials 116 and support factors 118 to form one or more bioinks. The generated bioink(s) are then used by a bioprinting device (e.g., bioprinter) 120 to print a 3D tissue 121 according to the 3D computer model, layer-by-layer (for multiple layers). The process then proceeds to 122 to grow tissue, in vitro , on the 3D tissue 121, and may further include disease modeling and/or screening using the printed 3D tissue 121. A final, 3D tissue (created by growing the tissue of the printed 3D tissue for a period of time in vitro ) 124 may then be applied in vivo at 126 (e.g., implanted into the target tissue/organ of the patient). For example, as shown at 126, the final 3D tissue may be a cardiac patch 128 for the heart of the subject.

As introduced above, there may be shortcomings to the bioprinting process, such as the bioprinting process described above with reference to FIG. 1. As one example, currently available printable materials (e.g., cells, biomaterials, support factors, and/or the like) may be limited, thereby limiting the bioprinted tissues to these materials and excluding the use of additional types of biomaterials for forming tissue constructs. As a result, the uses of the 3D printed tissue construct in the body or in certain disease modeling may be limited. Further, bioprinting cellular material may apply high shear levels to the cells, which may result in cell damage or stress. This may result in less viable tissue constructs for implanting in a patient or use in disease modeling. Further still, bioprinting an entire, large tissue structure, layer-by-layer using a bioprinter, for clinical applications may be time-consuming. This may increase the manufacturing costs and availability of these types of large 3D tissue constructs.

To address at least some of these issues, the inventors herein have recognized that a 3D vascularized tissue device may instead be comprised of a 3D vascular network formed by bioprinting with a sacrificial bioink that is removable (e.g., degradable) and a cast hydrogel (which is not bioprinted and contains cellular material, or cells, for forming tissue around the 3D vascular network). A 3D vascularized tissue device with this structure may allow for the device to be produced more rapidly with a desired vascular structure. Further, a greater variety of biomaterials for forming the tissue structure may be available, while applying lower shear stresses to the cells during formation of the device, due to casting the hydrogel within the perfusion chamber, around and within the bioprinted vascular network.

FIG. 2 shows a high-level schematic 200 of an embodiment of a process for generating a 3D vascularized tissue device, comprised of a bioprinted 3D vascular network and cast hydrogel, and perfusing the generated device using a bioreactor system. The final, printed construct formed via the process shown in FIG. 2 may comprise multiple types of tissues, such as tumor tissue that may be printed inside the 3D vascular network with a desired shape and geometry.

As shown in schematic 200, the process starts at 202 by bioprinting, with a bioprinter 204, the 3D vascular network 206 within a perfusion chamber 208. The 3D vascular network 206 may be formed via a sacrificial bioink (e.g., pluronic) which is adapted to be removed (e.g., degraded) later in the process. In some embodiments, during the bioprinting process, additional cellular structures may be printed within the 3D vascular network. For example, as shown in FIG. 2, selected tumor cells (e.g., neuroblastoma spheroids) are bioprinted, with a cellular, non- sacrificial, bioink, within the bioprinted 3D vascular network 206 to create a tumor 210 within a portion of the 3D vascular network 206. Thus, the bioink used to print the tumor 210 may be different than the bioink used to print the 3D vascular network 206 (e.g., may include the tumor cells instead of the sacrificial bioink). In one example, the 3D vascular network 206 and tumor 210 are simultaneously printed with the different bioinks. As shown in FIG. 2, the tumor 210 is printed into a central region of the 3D vascular network 206. However, in alternate

embodiments, the tumor 210 may be located within a different region of the 3D vascular network 206 (e.g., offset from the center). The tumor 210 may have a predetermined shape and geometry and printed according to these predetermined specifications (as defined by a user prior to printing, for example). In alternate embodiments, different types of cells may be bioprinted on, around, or in layers with the 3D vascular network, and/or different types of cells may be seeded into the printed 3D vascular network (and not bioprinted). Variations in these methods for different applications are described further herein.

At 212, the process includes casting a hydrogel (e.g., gelMA) 214 within and around the printed, 3D vascular network 206, within the perfusion chamber 208. The hydrogel 214 fills a majority of the perfusion chamber 208, at a level that is higher than the 3D vascular network 206. In this way, the 3D vascular network 206 is encased within the hydrogel 214. At 216, the hydrogel is cured via applied light (e.g., ultraviolet (UV) light or visible light) and the sacrificial bioink is removed via application of a selected fluid (e.g., PBS). As a result of this process, a 3D hollow vascular structure 218 is left behind (where the printed 3D vascular network previously resided), inside the hydrogel. The 3D hollow vascular structure 218 may be referred to herein as channels (e.g., microchannels) or vasculature. The resulting device is a 3D vascularized tissue device 220. A 2D view of the 3D vascularized tissue device 220 is shown at 222. As shown at 222, the channels of the 3D hollow vascular structure 218 are formed within the hydrogel 214. In the embodiment shown in FIG. 2, the hydrogel 214 forms a tumor microenvironment around the tumor 210.

At 224, the process includes perfusion of the 3D vascularized tissue device 220 within a bioreactor system 226. The bioreactor system 226 includes a series of 3D vascularized tissue devices 220 (arranged in series or in parallel with one another), each fluidly connected with a fluid unit 228 and media reservoir 230 and arranged within an incubator 231. The fluid units 228 are fluidly connected to a pump 232 which may be controlled by a flow control system 234. The environment of the incubator 231 may also be connected to a drying bottle 236 via air pressure tubing 238. Though four bioreactors (each including one 3D vascularized tissue device and associated fluid unit and media reservoir) are shown in the example of bioreactor system 226, a similar bioreactor system having more or less than four bioreactors is also possible. By using a bioreactor system with multiple 3D vascularized tissue devices 220, multiple devices 220 may be perfused at the same time. This may allow multiple 3D vascularized tissue constructs to be generated at the same time (thereby saving time in generating the tissue constructs) and/or the modeling or testing of multiple disease models or drugs at the same time. Further, as described further below, 3D vascularized tissue devices 220 which model different types of tissues (e.g., organs) may be used simultaneously within the same bioreactor system, thereby allowing the study of organ systems and their effects on different disease models or disease treatments (e.g., drugs).

In this way, FIG. 2 shows an example of a bioprinted, 3D vascularized tissue device, which is perfusable. As introduced above with reference to FIG. 2, the 3D vascularized tissue device includes a 3D vascular network comprising a sacrificial bioink, a hydrogel cast around the 3D vascular network, and, in some embodiments, additional bioprinted or manually seeded cells forming cell structures. As one example, the sacrificial bioink of the 3D vascular network may be comprised of non-crosslinked gelatins, pluronics, and/or other materials that may be easily removed upon application of an external fluid. For example, the sacrificial bioink may be soluble in water or another solution, such as phosphate-buffered saline (PBS). In some embodiments, the sacrificial bioink may degrade (or dissolve) when the 3D vascular network and/or entire printed 3D vascularized tissue device is submerged in and/or washed with the degrading fluid. In one example, the 3D vascular network may be comprised entirely of the sacrificial bioink, without any addition cellular material. In another example, a cellular material (such as endothelial cells) may be included within and/or combined with the sacrificial bioink and then used to print the 3D vascular network. For example, endothelial cells suspended in a sacrificial bioink material may be used as the bioink for printing the 3D vascular network.

FIGS. 17A-E illustrate an example of the viability of bioprinting 3D vascular networks with a sacrificial bioink containing human umbilical vein endothelial cells (FtUVECs). In particular, FIGS. 17A-D show microscopy images of endothelium formation onto printed microchannels (as indicated by arrows) and FIG. 17E shows a graph 1700 with relative growth (e.g., cell growth) on the y-axis and 3D cast constructs (used as a control) vs. the 3D printed constructs (e.g., the printed 3D vascular networks) for different culture time periods (e.g., 1, 7, and 14 days). As shown in FIGS. 17A-17E, the printed FtUVEC constructs were viable and proliferated under dynamic culture for 14 days.

As another example, the hydrogel that is cast within and around the bioprinted 3D vascular network may comprise gelatin methacrylate (gelMA), collagen, fibrin, and/or the like. The hydrogel, or hydrogel solution, may contain a variety of cells, small molecules (e.g., growth factors), nanoparticles, imaging contrast agents, and/or the like. The hydrogel may be crosslinked via the application of UV or visible light (e.g., depending on the grade of gelMA or alternate hydrogel material). In this way, the hydrogel may form the cellular tissue around (and of) the 3D vascular network. By casting the hydrogel around and within the 3D vascular network (instead of bioprinting the entire tissue construct), a larger variety of hydrogel solutions may be used (since the material is not limited to available printable materials). In some embodiments, in addition to the cells included in the liquid hydrogel solution, a variety of cells (such as endothelial cells) may be manually seeded into the printed 3D vascular network, hereby forming a cellular structure (e.g., endothelium) on the surface of the channels to better reproduce a vascular tissue structure (which is left behind after degrading the sacrificial bioink).

Turning now to FIG. 3, a flow chart of a method 300 for manufacturing a 3D

vascularized tissue device, such as the devices introduced above, is shown. Example

illustrations of the method for manufacturing the 3D vascularized tissue device are shown in FIGS. 4-12 and discussed below in conjunction with the discussion of method 300. FIGS. 4-12 include reference axes 420 including an x-axis, y-axis, and z-axis, for reference between figures and to relative dimensions.

Method 300 begins at 302 by generating a perfusion chamber and inlet and outlet channels into and out of the perfusion chamber with a desired geometry and dimensions. In one example, the perfusion chamber may be digitally designed and constructed via additive manufacturing (e.g., 3D printing) with a pre-defmed shape, geometry, and dimensions. For example, the perfusion chamber may be designed via computer aided design and then fabricated, according to the pre-defmed dimensions of the computer model, via additive manufacturing.

The shape, geometry, and/or dimensions of the perfusion chamber may be selected according to the intended use of the device, such as a type, size, and/or geometry of the tissue structure to be bioprinted within the perfusion chamber. In alternate embodiments, the perfusion chamber may be fabricated according to another technique, such as injection molding or another molding process. Additionally, the inlet and outlet channels connected to or included as part of the perfusion chamber may be shaped and sized to meet pre-determined perfusion requirements, such as a desired flow (e.g., perfusion) rate of fluid into and out of the perfusion chamber during perfusion of the tissue model within the device. As discussed further herein, the perfusion chamber may allow for real-time monitoring of flow through the engineering tissue construct (e.g., during perfusion, as discussed below with reference to the method at 320).

An embodiment of a perfusion chamber for the 3D, perfusable, vascularized tissue device is shown in FIG. 4. As shown in FIG. 4, a perfusion chamber 400 includes an inner chamber 402 depressed into an outer body 404 of the perfusion chamber 400. The perfusion chamber 400 may include an inlet channel 406 arranged within the outer body 404 and fluidly coupled to a first side of the inner chamber 402 and an outlet channel 408 arranged within the outer body 404 and fluidly coupled to an opposite, second side of the inner chamber 402. As shown in FIG. 4, an inlet 410 may be coupled to a first side of the outer body 404 and include an inlet flow passage 412 that mates with (and thus is continuously, fluidly coupled with) the inlet channel 406. Similarly, an outlet 414 may be coupled to a second side of the outer body 404 and include an outlet flow passage 416 that mates with (and thus is continuously, fluidly coupled with) the outlet channel 408. In one embodiment, as shown in FIG. 4, the inlet 410 and corresponding inlet flow passage 412, as well as the outlet 414 and corresponding outlet flow passage 416), are flared and increase in diameter as the inlet/outlet extends outward and away from the outer body 404. However, in alternate embodiments, alternate geometries, including diameters, of the inlet and outlets and their flow passages may be possible. For example, alternate embodiments for the inlet and outlets of the perfusion chamber are shown in FIGS. 14A-14B, as described further below. Further, in some embodiments, there may not be an inlet 410 and outlet 414 extending outward from the outer body 404 of the perfusion chamber 400. Instead, the inlets/outlets of the perfusion chamber may be included entirely within the outer body 404, as the inlet channel 406 and outlet channel 408.

Returning to FIG. 3, the method continues from 302 to 304 where the method includes casting a layer of hydrogel bioink into the bottom of the perfusion chamber and crosslinking the cast hydrogel layer. The hydrogel bioink (or hydrogel) may be comprised of any of the materials described above, such as gelMA. The cast layer of hydrogel may be a relatively thin layer relative to the overall height of the perfusion chamber. In one example, the thickness of the bottom layer of hydrogel may be approximately half the difference between the overall height of the perfusion chamber and the pre-determined height of the 3D bioprinted vascular network. In this way, the hydrogel within the perfusion chamber may fully surround the 3D bioprinted vascular network, as explained further below. Crosslinking the cast hydrogel layer may include exposing the cast hydrogel layer to light (e.g., ultraviolet (UV) or visible light) which may act to stabilize the bottom layer within the perfusion chamber. FIG. 5 shows an example of a layer of hydrogel cast into the bottom of the perfusion chamber 400. As shown in FIG. 5, a hydrogel layer 502 having thickness 504, in the direction of the y-axis, is cast into a bottom 506 of the inner chamber 402 of the perfusion chamber 400. The thickness 504 is smaller than a height 508, arranged in a direction of the y-axis, of the inner chamber 402 and the hydrogel layer 502 is arranged a distance below, relative to the bottom 506 and the y-axis, where the inlet channel 406 and outlet channel 408 connect to the inner chamber 402.

Returning to FIG. 3, method 300 continues from 304 to 306 where the method includes 3D bioprinting a 3D vascular network with a pre-determined architecture into the perfusion chamber. The pre-determined architecture of the 3D vascular network may include a pre- defined shape and geometry (e.g., design) which may be input to the bioprinter as a 3D computer model. The pre-determined architecture of the 3D vascular network may include a certain number of desired vascular channels and relative spacing between adjacent channels. In one embodiment, the pre-determined architecture may be determined based on clinical imaging data acquired from a patient. For example, the method at 306 may further include obtaining imaging data (e.g., 3D imaging data acquired from CT or MRI) of a target tissue of the patient, where the target tissue is the desired tissue to be mimicked or constructed with the 3D vascularized tissue device. Computer software may then be used to transform the acquired imaging data in a 3D computer model of the architecture of the 3D vascular network. This 3D model may then be used by the bioprinting device to print the 3D vascular network. In this way, patient-specific perfusable vascular tissue models may be created. This may be utilized for patient-specific disease modeling and/or drug screening and/or in the creation of tissues for tissue repair (such as cardiac patches), as discussed further below. The desired channels of the 3D vascular network are printed as a solid structure (e.g., negative of desired channel/vessel structure) using a sacrificial bioink. As described above and further below, the sacrificial bioink may be removable (e.g., degradable) further along in the process, leaving behind the desired architecture of 3D channels forming the vascular network within a cast hydrogel. Specifically, the method at 306 may include bioprinting, with the sacrificial bioink injected via a bioprinter, the 3D vascular network onto the cast layer of hydrogel at the bottom of the perfusion chamber. In some examples, the method at 306 may include setting printing parameters, prior to printing, the printing parameters including one or more of print speed, temperature, pressure, and needle size, all of which may be selected based on the pre-determined architecture and the intended application (e.g., type of tissue, perfusion conditions, clinical application, or the like). FIG. 6 shows an example of a 3D vascular network 602 printed into the perfusion chamber 400.

Specifically, the 3D vascular network 602 is printed using a bioprinting device 604 (which, in one example, may be an injection structure or needle of a bioprinter) onto the hydrogel layer 502, within the inner chamber 402 of the perfusion chamber 400. As shown in FIG. 6, the 3D vascular network 602 consists of a grid-like structure of solid (e.g., filled) channels 606 separated by spaces (e.g., voids) 608.

Following printing the 3D vascular network, at 308 of FIG. 3, method 300 includes inserting tubing into the inlet and outlet channels of the perfusion chamber and almost to the printed 3D vascular network. In one example, the inserted tubing may fully fill the spaces of the inlet and outlet channels. An example of tubing 702 inserted into the inlet channel 406 and outlet channel 408 of the perfusion chamber 400 is shown in FIG. 7. As shown in FIG. 7, ends of the tubing 702 extend all the way through the inlet and outlet channels, to the 3D vascular network 602.

Method 300 of FIG. 3 then continues to 310 to cast the remaining space, or a portion of the remaining space, within the perfusion chamber with a hydrogel. The hydrogel may be may be comprised of any of the materials described above, such as gelMA. Further, the hydrogel may be the same as the hydrogel layer cast into the bottom of the perfusion chamber prior to printing the 3D vascular network thereon. Casting the hydrogel into the perfusion chamber may include filling the interstitial space between and on top of the printed 3D vascular network of channels and fully encapsulating the entire 3D vascular network construct. The casting of the hydrogel at 310 may be performed as a single step, which is much faster than printing (e.g., bioprinting) the entire tissue construct of the 3D vascularized tissue device. For example, to bioprint the same thickness of tissue as is done by casting the hydrogel at 310, multiple layers of bioprinted cells and materials would need to be deposited, layer-by-layer. This layer-by-layer printing process takes significantly more time than casting the hydrogel layer as one step.

Following casting (in one example, immediately following) the hydrogel within the perfusion chamber, method 300 continues to 312 to crosslink (e.g., cure) the hydrogel using applied light. For example, the method at 312 may include applying UV or visible light, via an external light source in one example, to the device. In one embodiment, the wavelength of UV or visible light may be selected based on the grade or composition of the hydrogel. The light may be applied for a preset duration of time for crosslinking. In one example, the preset duration of time may be in a range of 1-2 minutes. In another example, the preset duration of time may be less than one minute. In yet another example, the preset duration of time may be less than two minutes. In one example, the preset duration of time may be chosen so that it is short enough (e.g., less than 1-2 minutes) to decrease the risk of partial degradation of the sacrificial ink within the hydrogel, which may result in a disturbed vascular structure. FIG. 8 shows an example of casting the hydrogel within the perfusion chamber 400, within and around an entirety of the 3D vascular network 602, and then crosslinking the cast hydrogel by applying light via a light source 802. As shown in FIG. 8, a second hydrogel layer 804 is cast into the inner chamber 402 of the perfusion chamber 400 via filling the inner chamber 402 with a liquid hydrogel solution that fills the inner chamber 402 and void spaces of the printed 3D vascular network 602. A height 806 of the second hydrogel layer 804 extends between a top of the bottom hydrogel layer 502 and a distance above a top surface (relative to the y-axis) of the printed 3D vascular network 602. The height 806 of the second hydrogel layer 804 plus a height of the bottom hydrogel layer 502 equal a total height 808 of hydrogel within the perfusion chamber 400. As shown in FIG. 8, the total height 808 of hydrogel is less than the height 508 of the inner chamber 402. However, in alternate embodiments, the total height 808 of the hydrogel may be equal to the height 508 of the inner chamber 402. In this way, the printed 3D vascular network is fully encased within the hydrogel. The light source 802 shown in FIG. 8 may be a UV light source or a visible (e.g., blue) light source and may be applied to the hydrogel for a pre-determined amount of time that may vary and be selected based on the composition of the hydrogel.

Continuing to 314 of method 300 in FIG. 3, the method includes removing the tubing (insert at 308) from the inlet and outlet channels of the perfusion chamber. Removing the tubing from the inlet and outlet channels may result in the formation of two hollow channels (e.g., microchannels) which connect the printed 3D vascular network structure to the orifices of the inlet and outlet channels. An example of the 3D vascularized tissue device with the tubing 702 removed from the inlet channel 406 and outlet channel 408 of the perfusion chamber is shown in FIG. 9. Removing the tubing results in the formation of two hollow channels 902 which each extend from a corresponding side of the 3D vascular network to an inlet orifice of the corresponding inlet or outlet channel to the inner chamber 402. As a result, after removing the sacrificial bioink, the hollow channels of the 3D vascular network may be perfused via fluid entering the inlet 410 of the perfusion chamber 400.

Method 300 of FIG. 3 then continues to 316 and includes degrading (e.g., removing) the sacrificial bioink of the printed 3D vascular network to create a 3D network of hollow vascular channels (also referred to herein as a 3D hollow vascular network). As one example, the method at 316 includes transferring the perfusion chamber to a container containing a degrading fluid adapted to degrade (e.g., dissolve) the sacrificial bioink of the printed 3D vascular network. For example, the degrading fluid may include a buffer solution (e.g., PBS, distilled water, or the like). The perfusion chamber may be submerged into the degrading fluid for a preset period of time until the sacrificial bioink is removed, leaving behind the 3D network of hollow vascular channels formed within the hydrogel. In alternate embodiments, the perfusion chamber may be washed with the degrading fluid, via flowing the degrading fluid through the device, until the sacrificial bioink is dissolved. FIG. 10 shows an example of submerging the entire perfusion chamber 400 within a container 1000 containing a degrading fluid 1002.

As shown at 318 in FIG. 3, method 300 may optionally include manually seeding a variety of cells into the hollow vascular channels. Manually seeding the cells may include using a manual injection device (not the bioprinter) to seed cells within an interior of the hollow vascular channels. As one example, endothelial cells may be seeded within the hollow vascular channels to better recapitulate the native vascular tissue structure. For example, the method at 318 may include seeding vascular cells (e.g., endothelial and/or smooth muscle cells) onto channel walls of the 3D network/structure of hollow vascular channels to form vascularized tissue channels. In one embodiment, the manual seeding of cells may be in addition to the option of adding cells to the liquid hydrogel solution cast into the perfusion chamber at 310.

The cells added to the liquid hydrogel solution to form the 3D tissue matrix may be different types of cells than vascular cells, in one embodiment.

Method 300 continues to 320 to connect the inlet and outlet channels of the perfusion chamber of the 3D vascularized tissue device to a bioreactor system and perfuse the tissue of the 3D vascularized tissue device. The bioreactor system may be similar to the bioreactor system shown in FIG. 2, as explained above. In one example, the method at 320 may include, via a bioreactor system, flowing various media (e.g., culture media, blood, drugs, and/or the like) into the inlet of the perfusion chamber, through the engineered vascular tissue (e.g., 3D network of hollow vascular channels), and out the outlet of the perfusion chamber, thereby perfusing the tissue of the 3D vascularized tissue device. Due to this perfusion, the tissue within the device may be grown further, preserved for implantation in a subject or for future testing, and/or used for disease modeling and/or drug testing. An example of the final 3D vascularized tissue device 1100, connected to a perfusion bioreactor system 1102, is shown in FIG. 11. Specifically, FIG.

11 shows the 3D network of hollow vascular channels 1104 formed within the cast hydrogel 804.

At 322, method 300 optionally includes removing the vascularized tissue construct (e.g., the tissue construct formed by the hydrogel and the 3D network of hollow vascular channels) from the perfusion chamber. For example, perfusing the 3D vascularized tissue device with blood and/or culture media may help to grow the vascularized tissue construct within the perfusion chamber into an engineered tissue that may be used for tissue repair or replacement in the body of a patient. As one example, as described further below, method 300 may be used to create a cardiac patch for tissue repair of a patient’s heart. Thus, at 322, the method may include removing the finalized cardiac patch, formed within the perfusion chamber, and then implanting the cardiac patch in a patient. An example of a vascularized tissue construct 1200 formed via method 300, after removal from the perfusion chamber or another container in which the construct is printed and cast, is shown in FIG. 12. Specifically, FIG. 12 shows the vascularized tissue construct 1200 including the 3D network of hollow vascular channels 1104 formed within a tissue structure 1202 (formed by culturing the hydrogel).

Method 300 then ends. However, in alternate embodiments, method 300 may additionally include closing a top of the perfusion chamber (e.g., via a slide, cap, or other structural feature) to fully cap/seal the inner chamber of the perfusion chamber and decrease a likelihood of dehydration of the tissue over a long-term culture period. In yet another embodiment, instead of closing the top of the perfusion chamber with a structural feature, the method may include filling the space above the cast hydrogel with a buffer solution or culture media to keep the tissue hydrated and also as a method to assess mass transport properties of the tissue (e.g., diffusion of reagents through the printed vasculature into the tissue and to the top of the inner chamber, or vice versa). The top cavity space of the inner chamber of the perfusion chamber may be further filled by casting and crosslinking additional hydrogel, which may help to ensure relatively leak-free perfusion.

As shown in FIGS. 6-12, the bioprinted 3D vascular network may comprise an array of overlapping and spaced apart channels (which are filled or solid before removal of the sacrificial bioink and hollow following removal of the sacrificial bioink). For example, as show in the embodiment of FIG. 12, the array of overlapping and spaced apart channels may include alternating rows of channels arranged in directions that are perpendicular to one another.

Specifically, as shown in FIG. 12, a first (e.g., top) row of channels may include a plurality of channels, spaced apart from one another, and all aligned in a first direction. Then, an adjacent, second row of channels may include a plurality of channels, spaced apart from one another, and all aligned in a second direction, the second direction perpendicular to the first direction. This pattern may repeat for the subsequent rows of channels. As seen in FIG. 12, void spaces are formed between the spaced apart channels in the same row and between channels in the directly adjacent rows (e.g., the rows above and below the current row). Channels in adjacent rows overlap one another, via the perpendicular arrangement of channels. In this way, the channels may be spaced apart in a lateral direction (either the z-axis or x-axis, depending on whether they are aligned in the direction of the x-axis or z-axis, respectively) and overlapping in a vertical direction (e.g., in the direction of the y-axis).

As discussed above, the spaces between adjacent channels of the 3D vascular network and 3D network of hollow vascular channels are filled by the hydrogel, and/or additionally bioprinted cell structures, which forms the tissue structure of the device. The 3D vascular network may be printed with various architectures. In one example, similar to the networks shown in FIGS. 6-12, the 3D vascular network may be printed in a crosshatch pattern with varying numbers of channels and spacing between the adjacent and overlapping channels. For example, the number of channels per layer and the spacing between adjacent channels of the same layer and the spacing between adjacent layers of channels may be selected based on the specific application (e.g., target tissue type and/or size) and/or imaging data of a subject.

Example images of a 3D vascularized tissue device having a crosshatch pattern for the 3D vascular network, and the 3D network of hollow vascular channels (after removing the sacrificial bioink), are shown in FIGS. 13A-13G.

Specifically, FIGS 13A-13C show images of 3D vascularized tissue devices including a bioprinted 3D vascular network 1300 within a perfusion chamber 1302. FIG. 13A shows the device prior to UV crosslinking of the hydrogel and FIG. 13B shows the device after UV crosslinking of the hydrogel. As seen in FIG. 13B, the vascular tissue constructs of the 3D vascularized tissue devices are semi-translucent, from at least the top, after UV crosslinking, allowing for real-time visualization of cellular growth and flow through the vascularized tissue. Visualization of flow through the device may allow for the use of different techniques, such as Particle image velocimetry, to quantify flow within the 3D vascular network.

FIG. 13C shows a fully assembled 3D vascularized tissue device with perfused vasculature. FIGS. 13D and 13E show another embodiment of a vascularized tissue device which is a standalone vasculature insert for biomimetic tissue (e.g., no perfusion chamber). FIG. 13D shows the device prior to UV crosslinking and FIG. 13E shows the device following UV crosslinking. Both FIGS. 13D and 13E shows the crosshatch pattern for the 3D vascular network. FIG. 13F shows a zoomed-in portion 1304 of the device from FIG. 13E. In particular, FIG. 13F shows the alternating pattern of hydrogel 1306 and vascular channel 1308 portions in each layer of the device. A zoomed-in view of one portion of a vascular channel 1310 (e.g., vessel track) is shown in FIG. 13G. In another example, the 3D vascular network may be printed in another pattern, other than the crosshatch pattern described above, such as in a series of parallel tubes/channels that are spaced apart from one another. In another example, the geometric structure of the 3D vascular network could be generated by 3D reconstruction of medical imaging data (e.g., CT or MRI) of a patient to create more biomimetic vascular tissues.

As introduced above with reference to FIG. 2 and FIG. 11, the 3D vascularized tissue device may be perfused within a bioreactor system, allowing for the growth and/or preservation of the tissue and/or the study of disease mechanisms/progressions and/or the effect of different drug treatments on diseased tissue. For example, by connecting the perfusion chamber of a 3D vascularized tissue device in a bioreactor system, either alone or in parallel or series with additional 3D vascularized tissue devices, the 3D vascularized tissue device may be perfused with a selected fluid and undergo various experiments and measurements.

Different embodiments of perfusion chambers for use in a 3D vascularized tissue device, for perfusion in a bioreactor system, are shown in FIGS. 14A-14G and FIGS. 15A-15C.

Specifically, FIGS. 14A-14G shows embodiments of a perfusion chamber for use with a solid organ tissue construct formed by the bioprinted 3D vascular network and cast hydrogel. A perfusion chamber may be generated with a combination of one or more features shown in the different embodiments of FIGS. 14A-14G. FIG. 14A shows a side view of a first embodiment of a perfusion chamber 1400 showing an inlet/outlet of the perfusion chamber having a tapered profile with a larger diameter inlet end 1402 and smaller diameter outlet end 1404, where the outlet end may fluidly couple to the inner chamber of the perfusion chamber. Thus, the inlet end 1402 and outlet end 1404 are spaced apart from one another, in a direction into the page. FIG. 14B shows an isometric side view of a second embodiment of a perfusion chamber 1406 showing an inlet/outlet of the perfusion chamber having a straight, non-tapered profile with an inlet end 1408 having a same diameter as the outlet end 1410 (though the diameters appear different in FIG. 14B, this is due to the isometric perspective of the figure and the inlet and outlet ends being spaced apart from one another, in a direction into the page). FIG. 14C shows a top view of a third embodiment of a perfusion chamber 1412 having a top/bottom 1414 that is open or transparent to allow for imaging of the tissue within the chamber. FIG. 14D shows a top view of a fourth embodiment of a perfusion chamber 1416 having a bottom 1418 that is closed and opaque or semi-transparent and adapted for long-term sterile perfusion. FIG. 14E shows an embodiment of an assembled device 1420 including an inlet (e.g., inlet barb) 1422 and outlet (e.g., outlet barb) 1424 directly and fluidly coupled to a perfusion chamber 1426. The tissue construct formed by the bioprinted 3D vascular network of hollow channels within the hydrogel may be arranged within an interior 1428 of the perfusion chamber 1426 and then sealed with a transparent top 1430, such as a microscope coverslip, as shown in FIG. 14F. FIG. 14F also shows an alternate set of inlet/outlets 1432 which are flared and not barbed. The bottom 1434 of the perfusion chamber 1426 in FIG. 14F is closed and semi-transparent, similar to the bottom shown in FIG. 14D. FIG. 14G shows the device of FIG. 14G, with a 3D vascularized tissue construct 1438 arranged therein and the perfusion chamber 1426 connected to fluid lines 1436 of a bioreactor system and undergoing perfusion. The design of the perfusion chamber, such as the top/bottom faces being transparent or semi-transparent, may allow for real-time monitoring of flow through the tissue of the devices.

FIGS. 15A-15C show an embodiment of a perfusion chamber 1500 for use with a cardiovascular or other vessel tissue construct for various flow studies. In particular, the perfusion chamber 1500 is a two-part device including a base 1502 to house the 3D vascularized tissue construct and a top 1504 to seal the construct within the base 1502. FIG. 15A shows a disassembled view of the two-part perfusion chamber 1500 while FIG. 15B shows an assembled view of the two-part perfusion chamber 1500 with the top 1504 directly coupled to the base 1502. As shown in FIGS. 15A-15C, the perfusion chamber 1500 is cylindrical in order to house a vessel-type tissue structure. In one embodiment, the top 1504 may be a removable, screw-cap for short to medium-term sterile perfusion. In another embodiment, the top 1504 may be permanently attached to the base 1502 via an adhesive for long-term sterile perfusion. As shown in FIG. 15C, the assembled perfusion chamber 1500 may be coupled with inlet/outlet barbs 1506. The inlet/outlet barbs 1506 may include helical protrusions 1508, forming barbed connections, for removably connecting each of the inlet/outlet barbs 1506 to fluid connections of a bioreactor system. The barbed connections may enable frequent connecting and disconnecting (in this way, being removably connected) of the 3D vascularized tissue devices to/from the bioreactor system, thereby allowing a variety of experiments and measurements to be performed with the devices.

In some embodiments, the perfusion chamber systems (also referred to as mini perfusion bioreactors), such as those shown in FIGS. 14A-G and FIGS. 15A-C may include and/or be fluidly coupled to a flow control valve for adjusting a flow rate of perfusion fluid from the bioreactor system to the 3D vascularized tissue device. In this way, the flow rate of fluid perfusing though the 3D vascularized tissue device may be controlled to a desired rate based on the application and/or adjusted during different flow studies. FIGS. 16A-16D show an embodiment of such a flow control valve 1600, including a sequence of assembly of the various parts of the flow control valve 1600. In particular, FIG. 16A shows a disassembled view showing the four main parts of the flow control valve 1600, including a valve body 1602, flow rate indicator (also referred to as a tracker) 1604, valve top cap 1606, and flow control knob 1608. The valve body 1602 includes fluid inlet/outlet connectors 1610, a main fluid passage 1612, a knob receiving chamber 1614 which is in fluid communication (e.g., fluidly coupled) with the main fluid passage 1612, and a bottom portion 1616 of a flow chamber 1620 (as shown in FIGS. 16C and 16D). The flow rate indicator 1604 is shaped as a wheel with a plurality of spokes that may rotate as flow travels through the main fluid passage 1612 and the flow chamber 1620. The valve top cap 1606 includes a top portion 1618 of the flow chamber 1620. Together, the top portion 1618 and the bottom portion 1616 of the flow chamber 1620 form the flow chamber 1620 when assembled, as shown in FIGS. 16C and 16D. The flow control knob 1608 includes a stem with a plurality of different sized flow openings 1622 arranged around a circumference of a distal end of the stem. The different sized flow openings 1622 allow for a larger range of achievable flow rates.

The sequence of assembly of the flow control valve 1600 may include first inserting the flow rate indicator 1604 into the bottom portion 1616 of the flow chamber 1620 within the valve body 1602, as shown in FIG. 16B. The valve top cap 1606 is then sealed onto the valve body 1602, as shown in FIG. 16C. As a result, the flow rate indicator 1604 is fully enclosed within the flow chamber 1620. Finally, as shown in FIG. 16D, the stem of the flow control knob 1608 is inserted into the knob receiving chamber 1614 of the valve body 1602. The flow control knob 1608 is rotatable within the knob receiving chamber 1614 in order to adjust the flow rate of fluid through the main fluid passage 1612, via the different sized flow openings 1622.

As introduced above, 3D vascularized tissue devices, such as those described above with reference to FIGS. 2-16, may be used in a variety of disease modeling and drug screening applications or for generating vascularized tissue constructs for tissue repair. For example, when used inside a perfusion chamber, the 3D vascularized tissue devices may be perfused with various fluids to study the mechanisms and progressions of various disease states and/or the effects of certain treatments (such as drugs or medications) on diseased tissue (e.g., the tissue generated within the 3D vascularized tissue device). In some embodiments, after appropriate cell culture/growth, the vascularized tissue constructs (formed by the 3D vascular network within the hydrogel) may be removed from the perfusion chamber or other vessel used to form the vascularized tissue, and then implanted into the body of a patient for tissue repair and/or replacement. Examples of such uses of the 3D vascularized tissue devices discussed above are detailed further below.

In one embodiment, the 3D vascularized tissue devices described herein may be used for cancer modeling and screening of cancer treatments, such as chemotherapy drugs. Cancer remains a leading cause of death, particularly among children. However, there are many constraints when attempting to study cancer causes, natural progression, and responses to therapy. Among these constraints in pediatric cancers are low incidences, substantial tumor heterogeneity and overlap with normal processes of rapid growth and developmental remodeling underway in children. Thus, there is a need to develop enhanced screening platforms to study novel molecular therapies for these diseases. While 2D cultures may allow for genetic and microenvironmental cue manipulation, they do not model the 3D architecture in which cancerous tumors reside. Animal models may recapitulate 3D architecture, but do not allow for facile genetic and microenvironmental perturbation. The 3D vascularized tissue devices discussed herein may provide 3D in vitro bioengineered tissue models that recapitulate the cellular, molecular, and microenvironmental complexity of tumors (such as pediatric and adult tumors) and allow for studying various cancer cell-small molecule interactions. For example, the 3D bioprinting and bioreactor technologies discussed herein may allow for precise spatial and temporal control of cells and biomaterials, as well as microenvironmental exposure of these tissues to variations in flow and oxygen perturbations. Specifically, by bioprinting cells and sacrificial bioinks at a relatively high spatial resolution (~20 pm), and casting hydrogels around the bioprinted vasculature, 3D vascular constructs may be created that can be perfused using a customized bioreactor system, such as the bioreactor system discussed above with reference to FIG. 2.

As discussed above, FIG. 2 presents an embodiment of a process for generating a 3D vascularized tissue device, comprised of a bioprinted 3D vascular network and cast hydrogel and perfusing the generated device. The process includes additionally bioprinting a tumor within the printed 3D vascular network and then casting the hydrogel around both the 3D vascular network and the tumor. The printed tumor may be a dense tumor spheroid. As one example, the printed tumor may consist of neuroblastoma (NB) cells in a hydrogel (e.g., gelMA) which are printed in the center of the 3D vascular network (e.g., 3D microchannel network containing endothelial cells). In particular, in one embodiment, a bioink used to print the NB tumor within the 3D vascular network may be prepared by initially growing NB cells in 2D culture media and then dissociating monolayers of NB cells into single cells and resuspending them in gelMA. To generate the 3D vascular network (e.g., microchannel networks), HUVECs may be suspended in a sacrificial bioink, as described above with reference to FIGS. 17A-17E. In one example, the FtUVEC bioink may be printed to form the 3D vasculature consisting of a number of layers (e.g., three), each layer containing 5 x 400pm-wide microchannels. NB bioink may be simultaneously printed in the center of the 3D vascular network (e.g., 600pm in diameter). Subsequently, microchanneled constructs may be crosslinked by UV radiation (e.g., 20 secs, 10 mW/cm2) and cultured in multiwell plates (static) for a threshold duration (e.g., four hours) to remove the sacrificial bioink and ensure cell attachment prior to culture with perfusion bioreactor, as discussed above with reference to FIG. 2 and further below.

NB is the most common extracranial pediatric cancer and NB patients often present with widespread metastases and despite aggressive therapies, less than half of them survive. As shown in FIG. 2 and described above, the resulting engineered vascular tissues containing the NB tumor (e.g., 3D vascularized tissue device) may then be perfused using a bioreactor system. In some examples, during perfusion, human vascular cells may be perfused through the 3D vascularized tissue device to form viable endothelium in the printed microchannels of the 3D vascular network. Over a period of culture and perfusion time of the 3D vascularized tissue devices containing the tumor(s), using the bioreactor system, cell viability and proliferation, expression of cell cycle/apoptosis related genes, as well as canonical NB drivers, and tumor tissue morphology, integration, and growth may be studied. In this way, the 3D vascularized tissue devices described herein may provide bioprinted tumor platforms that provide a highly biomimetic, vascularized microenvironment for the study of tumor biology and drug efficacy with significantly enhanced consistency and reproducibility.

The process described herein for creating 3D vascularized tissue devices may allow for the creation of spatially controlled 3D tumor architectures consisting of interconnected microchannel networks (e.g., vasculature) and a predefined distribution of cancer cells. In particular, 3D printing of microchannels within the tumor microenvironment, and their subsequent endothelialization, create a functional vascular network that allows for precise microenvironmental exposure of cancer cells to oxygen perturbations and various small molecules. The methods for generating and perfusing the 3D vascularized tissue devices discussed herein may allow for modeling of various tumor cell biology. Further, the bioprinting and hydrogel casting methods discussed herein may allow for the 3D vascularized tissue devices modeling various tumor microenvironments to be reproducibly printed in large numbers and then perfused in series/parallel configurations using a bioreactor system, thereby allowing for simultaneous, high-throughput screening of the effects of various developmental and/or microenvironmental cues on cancer cells. Further, the 3D vascularized tissue devices used for the bioprinted tumor platform may be cost-effective and facilitate manipulation of cell signaling and environmental cues, while also recapitulating the native tumor microenvironment.

Examples of studying disease progression and treatments in cancer applications using the 3D vascularized tissue devices (particularly those with the printed tumors) include studying the contribution of epithelial-mesenchymal transition (EMT)-activating transcription factors (EMT- TFs) to NB aggression (e.g., growth) under certain conditions (e.g., hypoxia) and/or treatments (e.g., chemotherapy). Further details on such studies using the 3D vascularized tissue devices as tumor models are included in ET.S. Provisional Application No. 62/682,363, filed June 8, 2018, which is incorporated herein by reference in its entirety.

As described above, various tumor tissues may be bioprinted inside 3D vascularized tissue devices and cultured under dynamic flow conditions using a bioreactor system. Various chemotherapy drugs may then be administered in the bioreactor reservoir (e.g., media reservoir 230 of FIG. 2) and perfused through the vascular tumor constructs (e.g., 3D vascularized tissue devices containing the tumor) to study drug interactions with cancer cells (e.g., specifically by investigating cancer cell viability, growth, aggression and metastasis by taking different measurements in vitro). In one example, chemoresi stance may be studied using these 3D vascularized tumor tissue device platforms. In another example, by using multiple devices, each mimicking different organs (as described further below with reference to multi-organ design and drug screening), the effects of cancer drugs on other tissues/organs (e.g., in addition to the cancer tissue) may be studied. For example, cardiotoxicity of cancer drugs may be studied using multi-organ design (e.g., cancer tissue - heart - liver) with multiple 3D vascularized tissue devices. In yet another example, the 3D vascularized tumor tissue device platforms may allow for the study of cancer metastasis in a highly biomimetic, dynamic environment. For example, healthy (non-cancerous) tissues within a first 3D vascularized tissue device may be connected to cancer tissue in a second 3D vascularized tissue device, within the same bioreactor system, and perfused with culture media for certain time periods. Cancer cell metastasis may then be evaluated and quantified by monitoring cancer cell migration from the cancerous construct into the healthy tissue.

In another embodiment, the 3D vascularized tissue devices described herein may be used for cardiovascular modeling and drug screening. For example, 3D vascularized tissue devices may be used as a platform for studying cardiac disease, including congenital heart defects. As one example, a perfusable cardiac tissue may be created using the methods for manufacturing 3D vascularized tissue devices disclosed herein in conjunction with human induced pluripotent stem cells (hiPSC), and their differentiation into specific cardiac cells including cardiomyocytes (heart muscle cells) and vascular cells such endothelial and smooth muscle cell. The perfusable cardiac tissue may then be cultured using a bioreactor system in vitro , and administered with a variety of small molecules or drugs to assess cell response. One example of this includes modeling coronary artery (vascular) diseases such as atherosclerosis (stenosis) using the 3D vascularized tissue devices. For this purpose, patient’s imaging (e.g., MR or CT) data may be used to reconstruct 3D models of the diseased vasculature, and the bioprinting process for the 3D vascularized tissue devices may be used to generate a patient-specific atherosclerosis model. Another example includes using the 3D vascularized tissue devices to study congenital heart diseases such as Hypoplastic Left Heart Syndrome (HLHS), using the patient’s medical imaging data to create personalized tissue models.

For example, FIG. 18 shows a high-level schematic 1800 of an embodiment of a process for generating a 3D vascularized tissue device to study HLHS. As shown in schematic 1800, the process starts at 1802 by obtaining hiPSC from HLHS donors 1804 and healthy donors 1806 (used as controls) and then differentiating the hiPSC colonies 1808 into hiPSC-cardiomyocytes (hiPSC-CMs) 1810 and hiPSC -endocardial cells (hiPSC-ECs) 1812. The hiPSC-CMs and hiPSC-ECs may then be incorporated into bioinks and bioprinted, along with bioprinting the 3D vascular network using a sacrificial bioink, at 1814, to create 3D vascularized cardiac tissue for studying HLHS. In alternate embodiments, the ECs may be manually seeded into the channels of the 3D vascular network to form the endocardium. A hydrogel may also be cast around the bioprinted vascular network and cardiac tissue to form the 3D vascularized tissue device 1816 for studying HLHS. The finalized bioprinted 3D vascularized cardiac tissue device(s) may then be perfused within a bioreactor system 1818 to study the disease mechanisms and progression of HLHS in response to different environmental factors.

FIGS. 19A-9B show example cross-sections of the bioprinted vascularized tissue environment of the 3D vascularized tissue devices created by the process of FIG. 18, for studying HLHS. In one embodiment, as shown in FIG. 19A, the bioprinted vascularized tissue environment 1900 may include a layer of endocardial cells 1902 on a Matrigel mattress substrate 1904 and the perfusion flow 1906 through the bioprinted vascular channels. In another embodiment, as shown in FIGS. 19B, the bioprinted vascularized tissue environment 1910 may additionally include a layer of cardiomyocytes 1912. In some embodiments, programmable bioreactor systems may be used to apply pre- defined flow regimens to perfused tissue models of the 3D vascularized tissue devices. As a result, the effect of flow hemodynamics on cellular/tissue function may be studied. This may be useful for vascular disease modeling, to simulate impaired/abnormal flow conditions in diseased/damaged vasculature.

In yet another embodiment, the 3D vascularized tissue devices described herein may be used for producing vascularized cardiac tissue patches (e.g., cardiac patches) for cardiac tissue repair. The cardiac patches may include a 3D construct of engineered cardiac tissue which is vascularized via a network of 3D hollow vascular channels within the tissue. In one

embodiment, a cardiac patch may be constructed via the method described above with reference to FIGS. 3-11. FIG. 20 shows a high-level schematic 2000 of another embodiment of a process for generating a cardiac patch for implantation in the body of a patient. As shown in schematic 2000, the process starts at 2002 by obtaining hiPSC-derived cardiomyocytes and suspending them within a hydrogel (e.g., gelMA) to form a bioink for printing. Then, at 2004, the derived bioink is transferred to a bioprinter 2008 for direct printing of the cardiomyocytes into a 3D cardiac tissue structure 2006, as shown at 2010. In one embodiment, the 3D cardiac tissue structure includes voids (e.g., spaces) between the printed cardiomyocytes for forming vasculature therein. For example, vascular-like cells (such as endocardial cells) may be directly seeded into these spaces or an additional sacrificial bioink including the vascular-like cells may be bioprinted with the bioprinter 2008, at the same time as printing the cardiomyocyte tissue structure, into the spaces and then removed later in the process, as described herein. In some embodiments, an additional hydrogel may be cast around the entire 3D cardiac tissue structure. At 2012, the process includes curing, via light (e.g., UV light) 2014 the printed cardiomyocytes (e.g., since they are suspended in a hydrogel). The resulting cardiac tissue patch 2016 may then be cultured, via a perfusion chamber and/or incubator, according to the perfusion methods described herein, until the cardiac patch has enough tissue growth and is ready to be implanted in the patient. FIG. 20 demonstrates a direct cell printing method to create the vascular (e.g., crosshatch) tissue structure. Thus, there may be no subsequent casting needed in this method. This is in contrast to the method described above with reference to FIGS. 3-11, which utilizes a “reverse engineering approach” where first, the vascular space is 3D printed and then the interstitial space between channels is filled (e.g., cast) by cells suspended in a hydrogel The direct printing method of FIG. 20 may be faster (as there would be no need for casting), however, it may introduce more shear stress to the cells (as compared to the method of FIGS. 3- 11) since they are directly printed (e.g., extruded) out of the print head (instead of being cast).

FIG. 21 shows a graph of cell viability and proliferation of endocardial cells (ECs) and cardiomyocytes (CMs) within a printed cardiac tissue patch, such as the cardiac tissue patch 2016 formed via the process shown in FIG. 20. Specifically, FIG. 21 shows a first plot 2100 of AlamarBlue assay reduction (%) indicating cell viability and proliferation of the ECs in the cardiac patch for a control patch produced via a traditional cell casting technique and the 3D bioprinted vascularized cardiac patch (produced via the process shown in FIG. 20) and a second plot 2110 of AlamarBlue assay reduction (%) indicating cell viability and proliferation of the CMs in the cardiac patch for the control patch produced via the traditional cell casting technique (both pre and post printing) and the 3D bioprinted vascularized cardiac patch. In the first plot 2100, AlamarBlue assay reduction is shown for days 1, 3, and 7 and for the second plot 2110, AlamarBlue assay reduction is shown for days 2, 5, and 8. As shown in both the first plot 2100 and the second plot 2110, the 3D bioprinted vascularized cardiac patch shows significant cell viability and proliferation by days 7 and 8 while the cell viability and proliferation tapers off by days 7 and 8 for the traditional cast cardiac patch. Thus, the method of producing the 3D vascularized cardiac tissue patches shown in FIG. 20 produce patches with viable cardiac tissue (both CMs and ECs) for implantation in a patient.

FIG. 22 shows graphs demonstrating the formation of functional CMs within the cardiac patch formed via the process shown in FIG. 20. Specifically, FIG. 22 shows a first plot 2200 of beating area (%) for the 3D bioprinted vascularized cardiac patch (e.g., produced via the method of FIG. 20) and the control (cast) patch, a second plot 2202 of beating rate (BPM) for the 3D bioprinted vascularized cardiac patch and the control (cast) patch, and third plot 2204 of deformation distance (mm) for the 3D bioprinted vascularized cardiac patch and the control (cast) patch. The 3D bioprinted vascularized cardiac patch shows a higher beating rate and deformation distance for the CMs than in the control patches and a relatively high beating area. Thus, the 3D bioprinted vascularized cardiac patch has functional CMs and may be implanted in a patient.

In a further embodiment, the 3D vascularized tissue devices described herein may be used for liver tissue modeling and drug screening for liver disease. Similarly to as described above for cardiac and cancer disease modeling and treatments, the 3D vascularized tissue devices described herein may be used to model liver tissue (healthy and/or diseased) and then be perfused within a bioreactor system with fluids including various treatments (e.g., drugs). Measurements may then be taken to examine the effects of the administered treatments on the diseased (or healthy) liver tissue. In some examples, drug metabolism by the modeled liver tissue may be studied under varying doses and/or flow conditions. The 3D vascularized liver tissue devices may also be perfused in combination with additional 3D vascularized tissue devices modeling other organs, in order to study multi-organ function, as described above and further below. In some embodiments, the 3D vascularized tissue devices may be generated to be patient-specific (e.g., according to patient imaging data and specific disease states).

FIGS. 23A-23F show example devices and metabolic activity for 3D vascularized tissue devices generated with liver tissue. Specifically, FIG. 23 A shows a graph 2300 of metabolic activity (fold increase) over a period of 14 days for bioprinted liver constructs that contain only liver cells (HepG2) and vascularized liver constructs that contain liver cells and endothelial cells (FtUVECs). As shown in FIG. 23 A, the endothelialized liver tissue constructs show increased metabolic activity compared to the liver-only constructs. FIG. 23B shows a graph 2302 of metabolic activity (fold increase) over a period of 14 days for bioprinted liver constructs that are perfused during that time (“Perfused liver construct”) and that are static and not perfused during that time (“Static liver construct”). As shown in FIG. 23B, the metabolic activity of the static liver constructs plateau by day 10 while the metabolic activity of the perfused liver constructs increases over the two-week period of perfusion. FIG. 23 C shows example vascularized liver tissue constructs (3D vascularized tissue devices) 2304, showing the endothelium 2306 formed within the liver tissue (which formed from the cast hydrogel) 2308. FIG. 23D shows one of the vascularized liver tissue constructs 2304 assembled within a perfusion chamber system 2310 for perfusion in a hybrid bioreactor. FIGS. 23E-23F show images of early (in first image 2312 of FIG. 23E) vs. late (in second image 2314 of FIG. 23F) endothelialization in bioprinted liver tissue constructs. In particular, FIGS. 23E-23F show compaction and remodeling of FtUVEC- based vascularization.

In still another embodiment, as introduced above, the 3D vascularized tissue devices described herein may be used for multi-organ design and drug screening. For example, the selection of materials and types of cells for the hydrogel of the 3D vascularized tissue device may be utilized to create vascular constructs mimicking various types of tissues/organs (e.g., heart, liver, kidney, lung, and the like). These 3D vascularized tissue devices mimicking different types of tissues/organs may be connected together via a bioreactor system and perfused in series with one another. This may allow for the study of tissue/organ function while interacting with other organs. Specifically, this may enable multi-organ drug screening, where potential side effects/toxicity of drugs on different organs/tissues may be simultaneously studied.

In yet another embodiment, the 3D vascularized tissue devices described herein may be used for various flow studies. For example, programmable bioreactor systems may be used to apply pre-defmed flow regimens to perfused tissue models and the effect of flow hemodynamics on cellular/tissue function may be studied. In one example, different flow rates of perfusion fluids may flow through the 3D vascularized tissue devices, at different times in the study period. Measurements of cell viability, proliferation, and the like, may then be taken to assess the flow effects on the tissue(s).

The above-described embodiments for use of the 3D vascularized tissue devices described herein are not meant to be limiting and other embodiments for the use of these devices may be possible. For example, the 3D vascularized tissue devices may be manufactured to contain/mimic tissue from any tissue in the human body and, as such, the material selection for the hydrogel and/or bioinks discussed herein may be adjusted to produce tissue of the desired type. While the 3D vascularized tissue devices are discussed herein as being produced and perfused within a perfusion chamber, theses 3D vascularized tissue constructs formed within the perfusion chamber may be removed from the perfusion chamber for implantation within a patient. Further, the 3D vascularized tissue devices may be formed as standalone vascularized tissue constructs on another suitable substrate or container, and not within a perfusion chamber.

In this way, a 3D vascularized tissue device may include a 3D network of vascularized channels formed within a tissue, formed via a combination of bioprinting a 3D vascular network and casting a hydrogel around and within the 3D vascular network. The bioprinted 3D vascular network may comprise a sacrificial bioink that is removed, after crosslinking the hydrogel within and around the 3D vascular network. Removing the sacrificial bioink of the 3D vascular network results in the creation of a 3D network of hollow vascular channels (e.g.,

microchannels) which are perfusable (e.g., fluid may flow through an interior of the channels).

In some embodiments, the bioink used to create the 3D vascular network may also contain vascular cell (e.g., endothelial cells), or vascular cells may be manually seeded (e.g., not bioprinted) within the channels of the 3D network of hollow vascular channels. As a result, the channels may be endothelialized and mimic vasculature within a tissue. The 3D tissue environment formed around the 3D network of hollow vascular channels is formed by the crosslinked hydrogel which may contain cellular material (e.g., cells or material for cell growth during perfusion/incubation). In some embodiments, additional cells may be bioprinted into/around portions of the 3D vascular network, at the same time as bioprinting the 3D vascular network. For example, this method may be used to form tumors or cardiac tissue within/around the 3D vascular network. The 3D vascularized tissue devices may include a perfusion chamber containing the tissue construct of the device. The perfusion chamber may be connected to a bioreactor system to enable perfusion of the device. This may facilitate tissue growth within the device and/or enable tissue disease modeling and drug screening studies with the devices.

The technical effect of manufacturing a three-dimensional (3D) vascularized tissue device via bioprinting a 3D vascular network, using a bioink material including a sacrificial bioink, within a perfusion chamber of the device; casting a hydrogel within and around the bioprinted 3D vascular network; and removing the sacrificial bioink, leaving behind a 3D structure of hollow vascular channels inside the hydrogel is the ability to generate a large variety of viable tissue constructs that have a desired vascular structure which is perfusable. For example, by forming the 3D vascularized tissue device via a combination of bioprinting the 3D vascular network and casting (and not bioprinting) a hydrogel around the printed 3D vascular network, the tissue constructs of the devices may be produced more quickly (as compared to bioprinting an entire tissue construct, layer-by-layer), in a reproducible manner, and without being limited to only available bioprintable materials. Additionally, by casting the hydrogel around and within the printed 3D vascular network, a 3D vascularized tissue construct may be created without applying higher levels of shear stress to the cells (as may be done via bioprinting). Further, the generation of a larger number of tissue types may be possible through the use of different hydrogel formulations. Further, the 3D vascularized tissue devices may allow for a variety of tissue disease model and drug screening studies, in an efficient manner, via connecting a plurality of devices in parallel with one another in a bioreactor system. Further still, by connecting a plurality of 3D vascularized tissue devices, mimicking different tissue types, in series with another in a bioreactor system, multi -organ studies may be conducted.

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. A three-dimensional (3D) vascularized tissue device, comprising:

a perfusion chamber;

a bioprinted, 3D vascular network comprised of sacrificial bioink, arranged within the perfusion chamber; and

a hydrogel cast within the perfusion chamber, around the 3D vascular network.

2. The device of claim 1, wherein the 3D vascular network is further comprised of cells bioprinted in combination with the sacrificial bioink.

3. The device of any one of the preceding claims, wherein the 3D vascular network comprises a series of layers of printed channels spaced apart in a lateral direction and

overlapping in a vertical direction, the lateral direction perpendicular to the vertical direction.

4. The device of claim 3, wherein the 3D vascular network is printed in a crosshatch pattern that define spaces between adjacently arranged printed channels of the 3D vascular network.

5. The device of any one of the preceding claims, wherein the hydrogel includes cells and is comprised of a material formulated to crosslink, upon an application of light, in less than two minutes and wherein the hydrogel is further cast into interstitial spaces of the 3D vascular network.

6. The device of any one of the preceding claims, wherein the perfusion chamber includes an inlet and an outlet, each including a barbed connection with helical protrusions for removably connecting the inlet and outlet to a bioreactor system.

7. The device of any one of the preceding claims, further comprising a 3D network of hollow vascular channels formed within the hydrogel via removing the sacrificial bioink, the hydrogel and 3D network of hollow vascular channels forming a tissue construct that is removable from the perfusion chamber.

8. A method for manufacturing a three-dimensional (3D) vascularized tissue device, comprising:

bioprinting a 3D vascular network, using a bioink material including a sacrificial bioink, within a perfusion chamber of the device;

casting a hydrogel within and around the bioprinted 3D vascular network; and removing the sacrificial bioink, leaving behind a 3D structure of hollow vascular channels inside the hydrogel.

9. The method of claim 8, further comprising, following casting the hydrogel, curing the hydrogel using light, the light including one or more of ultraviolet and visible light.

10. The method of claim 9, wherein curing the hydrogel using light includes applying a light source to the cast hydrogel for a preset duration of time, and wherein the preset duration of time is less than two minutes.

11. The method of any one of claims 8-10, further comprising, following removing the sacrificial bioink, seeding vascular cells onto channel walls of the 3D structure of hollow vascular channels to form vascularized tissue channels.

12. The method of any one of claims 8-11, wherein bioprinting the 3D vascular network includes bioprinting the 3D vascular network to have a predetermined 3D architecture based on acquired imaging data of a patient.

13. The method of any one of claims 8-12 wherein the bioink material further includes cells.

14. The method of any one of claims 8-13, further comprising connecting an inlet and outlet of the perfusion chamber to a bioreactor system and perfusing the 3D vascularized tissue device via circulating fluid through the 3D structure of hollow vascular channels.

15. The method of any one of claims 8-14, wherein casting the hydrogel within and around the bioprinted 3D vascular network includes casting the hydrogel all at once within the perfusion chamber at a thickness that encapsulates an entirety of the 3D vascular network.

16. The method of any one of claims 8-15, further comprising bioprinting a cellular, non- sacrificial, bioink into a region of the 3D vascular network at a same time as bioprinting the 3D vascular network to form a cellular structure.

17. A three-dimensional (3D) vascularized tissue device, comprising:

a 3D network of hollow vascular channels formed inside a cast and crosslinked 3D hydrogel via degrading sacrificial bioink of a bioprinted 3D vascular network,

wherein the hydrogel encapsulates the 3D network of hollow vascular channels and is interspersed in spaces formed between adjacent channels of the 3D network of hollow vascular channels.

18. The device of claim 17, wherein surfaces of channels of the 3D network of hollow vascular channels include endothelium formed by one or more of:

seeding vascular cells onto channels of the 3D network of hollow vascular channels; and including vascular cells with the sacrificial bioink used to bioprint the 3D vascular network.

19. The device of any one of claims 17 and 18, wherein the hydrogel includes one or more of gelatin methacrylate, collagen, and fibrin and wherein the hydrogel further includes cells, the hydrogel forming a 3D tissue structure around the 3D network of hollow vascular channels.

20. The device of any one of claims 17-19, further comprising a perfusion chamber and a chamber inlet and outlet, wherein the 3D network of hollow vascular channels and hydrogel are formed within and removable from an inner chamber of the perfusion chamber.