Method and system for multi-cell bioprinting vascularized tissue constructs

Abstract

The present invention relates to systems, compositions and methods for multicellular bioprinting vascularized tissue constructs comprising a hierarchical vascular network. The methods employ a dual bio-ink strategy involving fibrous microgel matrix bio-inks with adjustable hardness, self-healing rheology, and microporosity to support capillary self-assembly, and sacrificial bio-inks that define a large pourable channel after removal. The system integrates top-down printing and bottom-up cell self-organization within a programmable bio-printing platform. The key aspect of the invention is to engineer the physical topography of the matrix to actively guide vascular morphogenesis beyond conventional permissive stents. The integrated platform enables the manufacture of complex bionic vascular networks with enhanced mass delivery, cellular viability, and in vivo integration potential. The invention represents an important progress in the aspect of extendable production of functional human tissues, and has application in regenerative medicine, disease modeling and drug discovery.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 722,558, filed November 19, 2024, and U.S. Patent Application No. 19 / 369,313, filed October 26, 2025, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention relates in general to at least the fields of regenerative medicine and tissue engineering, bioprinting technology, and drug discovery. Background Technology

[0003] Engineered tissues with functional, multi-scale vascular networks are crucial for addressing major challenges in regenerative medicine. In natural organs, hierarchical vascular systems composed of macrovessels, microvessels, and capillaries ensure efficient oxygen and nutrient transport, waste removal, and dynamic signal transduction. Without such networks, tissue constructs exceeding several hundred micrometers in thickness often suffer from hypoxia, leading to necrosis and loss of function. To reconstruct these physiological characteristics, engineered tissues must combine perfusionable macrovascular structures and capillary-like microvessels in a spatially organized and scalable manner. These multi-scale systems are essential not only for transplantable tissue grafts but also for in vitro platforms used in drug discovery, ischemia modeling, and research on organ-specific diseases.

[0004] Existing vascularization strategies are generally categorized into two types: top-down and bottom-up approaches. Top-down techniques, such as extrusion-based 3D bioprinting, allow for the precise patterning of perfusionable channels and are effective for replicating macroscopic structures. However, the use of high-concentration bulk hydrogels often restricts cell movement and matrix remodeling, thereby impairing the formation of microvascular systems that mimic native tissue. Bottom-up approaches, such as organoid formation or self-assembly within permissible hydrogels, support microvascular development but typically lack structural control, scalability, and perfusion.

[0005] Current 3D-printed vascular models derived from imaging data can faithfully replicate the geometry of large blood vessels, but they cannot reproduce capillary budding or the complexity of microvessels. Conversely, vascular organoids provide microvascular mimicry, but are embedded in amorphous matrices such as Matrigel, which lack mechanical strength and are incompatible with scalable tissue fabrication. These limitations are particularly pronounced in liver tissue engineering, where current artificial constructs often fail to replicate the sinusoidal structure of the natural hepatic vascular system. Consequently, hydrogels loaded with hepatocytes frequently exhibit poor perfusion, central necrosis, and short-lived survival.

[0006] Several prior art references illustrate some progress in this field, but none fully address the challenges of scalable multi-scale vascularization. For example, U.S. Patent Application Publication No. US20200289709 A1 discloses tissue constructs using an undefined “microparticle-like tissue” and a general “ink” formulation, but does not describe a dual bio-ink strategy or specific method for forming hierarchical vascular structures. While the literature mentions perfusionable networks, it lacks a clear description of the microvascular and capillary generation mechanisms and provides no in vivo assessments to demonstrate their integration potential. Similarly, U.S. Patent Application Publication No. US20180313822 A1 focuses on engineered tissues for in vitro use and describes pseudovascular networks, but does not emphasize multi-scale vascular structures or provide methods for combining macrovessels and microvessels in a coherent system. Furthermore, the methods are limited to an in vitro context and do not imply host integration or in vivo applicability. Despite these advances, no strategy currently exists that successfully integrates architectural precision and microvascular self-organization into a single scalable platform. Therefore, current techniques remain insufficient to encompass the structural and functional complexity of naturally vascularized tissues.

[0007] Therefore, there is a need in the art for a unified vascularization strategy that can scalably form multi-scale, perfusion, and tissue-specific vascular networks, overcoming the structural and functional limitations of current methods. Summary of the Invention

[0008] This invention aims to address a fundamental challenge in tissue engineering: the scalable production of thick, vascularized tissues that realistically reproduce the hierarchical vascular structure of natural organs. Specifically, this invention seeks to overcome the limitations of existing top-down and bottom-up biomanufacturing methods, which have so far failed to provide an integrated platform capable of generating perfusionable macrovascular channels and bioorganic microvascular networks within a single tissue construct.

[0009] This invention provides an integrated biomanufacturing platform for fabricating engineered tissue constructs containing hierarchical vascular networks. It overcomes a fundamental limitation in tissue engineering: the inability to generate thick, metabolically active tissues, including perfusionable macroscale channels and biologically relevant microvascular structures. This challenge is overcome through a synergistic combination of novel fibrous microgel bioinks and a variety of advanced bioprinting structures.

[0010] The fibrous microgel bioink described in this invention possesses a unique set of properties designed to support angiogenesis and enable complex tissue fabrication. First, it comprises a microporous structure formed by discrete microunits, creating a highly permeable network that supports mass transport and cell migration. Second, it exhibits thixotropy and self-healing rheology, enabling the bioink to serve both as a printable matrix and as a support bath for embedded or suspension printing. Third, its mechanical stiffness can be tuned via photopolymerization to match the stiffness of natural tissues (e.g., liver). Fourth, and most critically, the microscopic morphological features of the fibrous network provide structural cues guiding vascular cells to self-organize into tissue-specific capillary structures, such as sinusoidal networks. These capabilities distinguish the fibrous microgel from conventional bulk hydrogels, which lack permeability, responsiveness, or biomimetic structures.

[0011] The method employs a dual bio-ink strategy, wherein a matrix bio-ink (e.g., a fibrous microgel with encapsulated cells) is printed together with a sacrificial bio-ink, which can be selectively removed to form open, permeable large channels. In some embodiments, the sacrificial ink may be cell-loaded and have a time delay upon dissolution, allowing released endothelial cells to adhere to the channel surface and form a luminal liner. In this way, the present invention integrates structural precision with bio-self-organization into a single manufacturing workflow.

[0012] In one aspect, the present invention provides a method for multicellular bioprinting of vascularized tissue constructs, the method comprising: preparing a biodegradable matrix bioink comprising gelatin methacrylate (GelMA) and gelatin; preparing a sacrificial bioink comprising a hydrogel-forming component and a calcium agent; loading the matrix bioink and the sacrificial bioink into separate cartridges of a multi-nozzle 3D bioprinter; co-printing the matrix bioink and the sacrificial bioink in alternating or embedded patterns to form a 3D tissue construct; crosslinking the matrix bioink and removing the sacrificial bioink to create perfusion channels in the construct; and forming the vascularized tissue construct comprising an integrated network of a biodegradable matrix and perfusion channels.

[0013] According to one embodiment, the matrix bioink further comprises alginate.

[0014] According to one embodiment, the hydrogel-forming component includes gelatin, hyaluronic acid, agarose, alginate, gellan gum, collagen, polyethylene glycol (PEG)-based polymers, or combinations thereof.

[0015] According to one embodiment, the calcium agent includes calcium chloride, calcium sulfate, or a combination thereof.

[0016] According to one embodiment, the matrix bio-ink comprises microporous fibrous microgel particles formed by splitting cross-linked bulk hydrogels, and the fibrous microgel particles are prepared by extruding the cross-linked hydrogel through a 20 μm sieve.

[0017] According to one embodiment, the sacrificial bio-ink comprises inks with a density of 5 × 10⁻⁶. 5 10 cells / mL to 2×10 6 Endothelial cells in the range of cells / mL, wherein the cells adhere to the surface of the inner channel after sacrificial ink removal.

[0018] According to one embodiment, the three-dimensional tissue construct includes a hexagonal large-channel geometry that simulates the natural liver lobule.

[0019] According to one embodiment, the sacrificial bio-ink is directly printed into a self-healing support bath comprising a microporous matrix blocked by fibrous microgels. The support bath self-heals around the printed filaments prior to crosslinking and contains a first cell population, such that the resulting three-dimensional tissue construct exhibits a hierarchical porosity defined by the printed large channels and inherent microporosity, and supports capillary self-assembly within the matrix.

[0020] According to one embodiment, the printed large channel defines a fillable circuit, the fillable circuit including at least one inlet and at least one outlet.

[0021] According to one embodiment, the sacrificial bio-ink is removed after printing by thermal liquefaction or enzymatic degradation.

[0022] According to one embodiment, the crosslinking step is performed by photocrosslinking and / or ionic crosslinking.

[0023] In another aspect, the present invention provides a vascularized tissue construct for implantation into a host organ. The vascularized tissue construct comprises a microporous matrix having interconnected micropores formed from a bio-ink composition; a perfusionable network of macrochannels extending through the microporous matrix; and one or more populations of living cells encapsulated within the microporous matrix. The vascularized tissue construct exhibits hierarchical porosity defined by the macrochannels and the interconnected micropores of the microporous matrix. The construct includes a self-organized microvascular plexus formed within the microporous matrix.

[0024] According to one embodiment, the bio-ink is formed by: (a) forming a pre-gel solution comprising GelMA, gelatin, and LAP; (b) photocrosslinking the solution to form a bulk hydrogel; (c) splitting the hydrogel through a sieve to form microparticles; (d) suspending the microparticles in a phosphate-buffered saline solution; and (e) centrifuging the suspension at 10,000 rpm for 5 minutes to remove excess liquid, thereby obtaining the final printable bio-ink.

[0025] According to one embodiment, the bioink composition comprises a suspension of cross-linked hydrogel microparticles containing GelMA and gelatin, wherein the suspension of cross-linked hydrogel microparticles forms a blockage microporous matrix having interconnected pores in the range of 20 μm to 50 μm, and the matrix exhibits shear-thinning and self-healing thixotropic properties, and provides morphology guidance to support the formation of tissue-specific structures.

[0026] According to one embodiment, the interconnected micropores are configured to guide vascular cells to form sinusoidal capillary structures.

[0027] According to one embodiment, the inner surface of the large channel is lined with endothelial cells derived from sacrificial ink.

[0028] According to one embodiment, the encapsulated cells comprise a co-culture of human mesenchymal stem cells and endothelial cells, wherein the endothelial cells, guided by the morphological features of the matrix, self-organize into branched capillary plexuses along the interstitial spaces of the microporous matrix.

[0029] According to one embodiment, the large-channel perfusionable network includes at least one inlet and at least one outlet, the at least one inlet and the at least one outlet being configured to allow bidirectional perfusion through the tissue construct.

[0030] According to one embodiment, the microporous matrix simulates the microstructure of the liver sinusoidal space.

[0031] In another aspect, the present invention provides a system for bioprinting vascularized tissue constructs. The system includes: a programmable multi-nozzle 3D bioprinter having at least two independently controlled printheads; one or more temperature-controlled cartridge dispensers for bio-ink; a photocrosslinking device configured to deliver blue light (405 nm); and a chamber for containing a self-healing support bath during embedded or levitation printing. At least one temperature-controlled cartridge dispenser is configured to dispense gelatin-based sacrificial bio-ink at 20-30°C.

[0032] According to one embodiment, the system further includes a dual-syringe Y-connector for mixing a cell suspension into a matrix bio-ink under aseptic conditions.

[0033] According to one embodiment, the system further includes nozzles of different sizes for generating vascular structures with different channel diameters.

[0034] According to one embodiment, the at least one independently controlled printhead is pneumatically pressure driven and digitally controlled to regulate extrusion.

[0035] According to one embodiment, the system further includes a microfiber bioink preparation device, which includes a nylon screen (e.g., a 20.0 μm nylon membrane filter) with a pore size of approximately 20.0 μm and a planetary centrifugal mixer.

[0036] According to one embodiment, the system further includes a PDMS chamber configured to contain the self-healing support bath during levitation printing.

[0037] Compared to conventional methods that rely solely on large-channel printing or microvascular assembly, this invention implements both strategies in a coordinated and reproducible manner. The platform supports modularity across tissue types, compatibility with various bio-ink components and cell sources, and scalability for larger constructs. Experimental results demonstrate enhanced nutrient diffusion, high cell viability, formation of endothelial-lining vessels, and preliminary in vivo integration. Applications include regenerative medicine, organ-specific disease models, and high-throughput drug testing using physiologically relevant vascularized tissues.

[0038] Key innovations of this invention include a dual bio-ink strategy, multi-scale vascularization, and a programmable multi-nozzle bioprinting implementation. Specifically, the method employs a matrix bio-ink composed of gelatin, alginate, and gelatin methacrylate (GelMA) and a sacrificial bio-ink containing hyaluronic acid, gelatin, and calcium, optionally loaded with endothelial cells. This dual system enables precise spatial control over cell distribution and the formation of microcapillary networks and perfusion-grade macrochannels. The matrix bio-ink undergoes in-situ cross-linking via visible light and calcium ion exchange, enhancing construct integrity while maintaining microporosity. The fibrous microgel matrix exhibits thixotropic and self-healing properties, supporting various printing formats, including staggered, embedded, and suspension printing. Furthermore, the incorporation of a 1:1 co-culture system of human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (hMSCs) enhances endothelial budding and lumen formation, while the delayed sacrificial ink based on 7.5% gelatin promotes the gradual formation of large endothelial-lined vascular channels. Preliminary in vivo results demonstrate promising integration potential, including host cell infiltration and expression of human-specific vascular markers. Overall, these features establish a versatile and scalable bioprinting platform capable of generating physiologically relevant vascularized tissue constructs for applications in regenerative medicine, disease modeling, and drug discovery. Attached Figure Description

[0039] Embodiments of the invention are described in more detail below with reference to the accompanying drawings, in which:

[0040] Figure 1A-1B A schematic illustration shows a multicellular bioprinting method for tissue constructs. Figure 1A Illustration of sacrificial bioprinting based on direct ink writing (DIW): staggered printing of a biodegradable matrix and sacrificial bio-ink. Removal of the sacrificial material after incubation creates interconnecting channels. Figure 1B Multiscale vascularization approach: a combination of microfiber bioink (using human mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs)) for capillary networks and sacrificial bioink (using HUVECs) for larger vessels.

[0041] Figure 2A Confocal micrographs comparing the nanoporous structure of conventional bulk hydrogels with the interconnected microporous network of fibrous microgels are shown. Scale bar = 200 μm. Figure 2B A rheological evaluation is presented, which demonstrates the thixotropic and self-healing properties of the fibrous microgels under alternating high (100%) and low (1%) strain conditions, compared to the stable solid-like response exhibited by conventional bulk hydrogels. Figure 2C The images shown over time demonstrate the rapid permeation of 2000 kDa fluorescent dextran (blue) through the fibrous microgel, while conventional bulk hydrogels remain impermeable, thus proving the superior mass delivery properties of the fibrous microgel.

[0042] Figures 3A-3C Degradation analysis of the construct printed with dual bio-inks is shown. Figure 3A Images of the printed construct degrading within 10 days. Scale bar: 5mm. Figure 3B The remaining weight of the printed constructs before and after soaking in PBS with different concentrations of calcium chloride. Figure 3C ) 10-day degradation rate of constructs with different calcium concentrations.

[0043] Figure 4 A graph depicting the compressive modulus of fibrous microgels (MF) and matrix bulk hydrogels (Bulk) after photocrosslinking with 405 nm light for different durations is presented. The compressive modulus of native mouse liver is included as a physiological benchmark for comparison.

[0044] Figures 5A-5B A dual-bio-ink method for forming void channels in 3D bioprinted constructs is illustrated. Figure 5AA schematic diagram of an in-situ crosslinking method for bioprinting substrates and sacrificial inks. Figure 5B (i) Comparison of print fidelity: (i) Lattice structures printed with no calcium and (ii) with 1% calcium. Scale bar: 1 mm. (iii) Small-scale printed constructs. Scale bar: 5 mm. (iv) Forming void channels using nozzle #25. Scale bar: 400 μm. (v-viii) Forming staggered structures and void channels using nozzle #30. Scale bar: (v and vii) 1 mm, (vi and viii) 400 μm.

[0045] Figure 6A A conceptual schematic diagram is shown illustrating how multimaterial bioprinting, using a matrix and sacrificial bioink, can produce porous tissue constructs with pre-designed heterogeneous cellular tissues after incubation. Figure 6B A schematic diagram (top) and a corresponding optical microscope image (bottom) detailing the layer-by-layer fabrication process of a bioprinted construct are shown. The workflow demonstrates an orthogonal stacking of interwoven filaments consisting of a matrix and sacrificial bio-ink, followed by the removal of the sacrificial material to produce a porous matrix scaffold.

[0046] Figure 7A A macroscopic view of a printed construct loaded with hepatocytes is shown. Figure 7B The image shows an optical microscope image of the microstructure and cellular tissue within the printed construct. Figure 7C Confocal microscopy images show the spatial patterns and interactions of multiple cell types in a bioprinted construct. Figure 7D A confocal image of a co-culture construct containing human mesenchymal stem cells (hMSCs, green) and human umbilical vein endothelial cells (HUVECs, dark red) is shown. Scale bar = 200 μm.

[0047] Figure 8A A schematic diagram illustrating a subcapsular liver implantation procedure for in vivo evaluation of bioprinted constructs. Figure 8B A gross view of the explanted liver seven days post-implantation is shown, in which the bioprinted hydrogel construct is clearly retained at the surgical site (marked with dashed lines). Figure 8C The images show contrasting hematoxylin and eosin (H&E) staining of liver tissue seven days post-implantation. (i) The cell-free block hydrogel control shows a clear boundary with the host tissue and no cell infiltration. (ii) The cell-loaded printed construct shows extensive host blood cell infiltration (indicated by black arrows) and seamless integration with the surrounding liver tissue. Figure 8D Immunohistochemical analysis of the cell-loaded bioprinted constructs is shown. (i) Human CD31-positive endothelial structures were detected and indicated by black arrows. (ii) Human CD133-positive cells were similarly identified by black arrows. Scale bar = 200 μm.

[0048] Figure 9A A schematic illustration of the stacked hexagonal lobule structure of a natural liver is shown, which serves as a biomimetic design inspiration for vascularized tissue constructs. Figure 9B An embedded printing strategy is illustrated, comprising (A) a microfiber matrix bio-ink, (B) a sacrificial ink patterned into repeating leaf-like units, and (A+B) a process of embedding the sacrificial ink into the matrix to produce a biomimetic hexagonal structure. Figure 9C The final printed construct containing an embedded sacrificial network is shown. The illustration shows an enlarged schematic of a single repeating unit after the sacrificial material has been removed, revealing a triangular tissue region surrounded by interconnected infusible channels.

[0049] Figure 10A An illustration shows an experimental setup for perfusion using bioprinted vascular channels constructed in vivo. Figure 10B The diagram illustrates the concept of multi-scale vascularization achieved through suspension bioprinting, which integrates large, perfusionable macrovascular channels with a self-assembled microvascular network formed within a fibrous matrix.

[0050] Figure 11A A schematic illustration of the in situ vascularization process is shown, depicting the continuous process from the initial deposition of endothelial cells along the channel wall to the formation of budding vascular structures that invade the surrounding matrix. Figure 11B Fluorescent images of HUVEC behavior in the bioprinted construct are shown. (i) Initial cell deposition and aggregation were observed along the channel walls on day 1. (ii) By day 7, HUVECs exhibited significant proliferation and invasion of the surrounding matrix, forming elongated vascular shoots. Scale bar = 200 μm.

[0051] Figure 12A This illustrates the formation of a broad, interconnected capillary network within the fibrous microgel on day 10, resulting from the co-culture of endothelial cells and mesenchymal stem cells. Figure 12B The maturation of the capillary network on day 14 is shown, forming distinct hollow lumens (indicated by asterisks), representing a key structural feature of the functional microvascular system. Scale bar = 50 μm.

[0052] Figures 13A-13D A dual-bio-ink method for bioprinting multicellular tissue constructs is illustrated. Figure 13A Cell viability in matrix bioinks: (i) live / dead staining images at different cell densities; (ii) quantitative cell viability analysis. Figure 13B Post-printing after sacrificial ink removal (i-ii). Dense tissue-like structures formed after 48 hours (iii). Scale bar: 200 μm. Figure 13C Multicellular printed constructs: (i-iii) staggered printing patterns of matrix (M) and sacrificial (S2, S1) bio-inks, scale bar 5 mm; (iv-viii) different types of cell tissue in the printed constructs. Figure 13D Cells released from the sacrificial ink adhere to the matrix surface, forming void channels within the printed construct. Scale bar: 200 μm.

[0053] Figure 14A A structural comparison based on H&E staining of mouse liver tissue is shown, revealing the native sinusoidal septum structure. This structure exhibits a network of interstitial voids resembling a network of fibers spatially arranged within a fibrous microgel. Figure 14B The diagram illustrates a guided self-assembly process in which co-cultured HUVECs and hMSCs utilize fibrous microgel structures as structural templates to form an endothelialized network within the interstitial spaces. Figure 14C Confocal micrographs of F-actin and DAPI staining after 14 days of culture are shown, revealing that the cells have self-organized into a continuous and interconnected network that mimics the structure of the hepatic sinusoids, demonstrating morphological guidance provided by fibrous microgels.

[0054] Figures 15A-15D A morphological comparison of cells cultured in bulk and microfiber hydrogels is shown. Figures 15A-15B Cells encapsulated in a block-shaped hydrogel. Figure 15C-15D Cells loaded in a microfiber hydrogel. Scale bar: 100 μm.

[0055] Figure 16 The time course of HUVEC monoculture and HUVEC-hMSC coculture in microfiber hydrogels is shown as a comparison over 7 days. Scale bar: 50 μm.

[0056] Figure 17 Extended cultures of HUVEC monocultures and HUVEC-hMSC cocultures in microfiber hydrogels are shown on days 10 and 14. Lumens are marked with an asterisk (*). Scale bar: 50 μm.

[0057] Figures 18A-18E The three-dimensional bioprinting and development of large blood vessel structures using sacrificial bio-ink loaded with HUVECs is demonstrated. Figures 18A-18B The 3D-printed constructs demonstrate the scalability and reproducibility of the dual bio-ink hydrogel platform. Scale bar: 1 cm. Figure 18C The distribution of HUVECs within the sacrificial ink channels on day 1 illustrates the initial cell aggregation. Figure 18D On day 7, sacrificial ink channels and surrounding HUVEC tissue showed cell invasion of the surrounding matrix. Figure 18E F-actin staining revealed the budding vascular structure and interstitial space, indicating angiogenesis. EG scale bar: 200 μm. Detailed Implementation

[0058] definition

[0059] Throughout this specification, unless the context otherwise requires, the word “comprise” or variations thereof, such as “comprises” or “comprising”, should be understood to imply inclusion of the stated whole or group of wholes, but not to exclude any other whole or group of wholes. It should also be noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as “comprises,” “comprised,” and “comprising” may have the meaning attributed to them under U.S. patent law, for example, allowing for elements not expressly listed but excluding elements present in the prior art or affecting the essential or novel features of the invention.

[0060] Furthermore, throughout this specification and claims, unless the context otherwise requires, the word "include" or variations such as "includes" or "including" shall be understood to imply inclusion of the stated whole or group of wholes, but not to exclude any other whole or group of wholes.

[0061] As used herein and unless otherwise defined, the terms “substantially,” “basically,” “approximately,” and “about” are used to describe and explain small variations. When used in conjunction with an event or situation, the terms may cover instances where the event or situation occurred precisely or instances where the event or situation was close to occurring. For example, when used in conjunction with a numerical value, the terms may cover a range of variation less than or equal to ±10% of the numerical value, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%.

[0062] References to "an embodiment," "an example embodiment," "exemplary embodiment," etc., in this specification indicate that the described embodiment may include specific features, structures, or characteristics; however, not every embodiment necessarily includes specific features, structures, or characteristics. Furthermore, such phrases do not necessarily refer to the same embodiment. Moreover, when a specific feature, structure, or characteristic is described in connection with an embodiment, whether explicitly described or not, it is assumed that the influence of such feature, structure, or characteristic on other embodiments is within the knowledge of those skilled in the art.

[0063] "Matrix bioink" refers to a bioink formulation containing at least one structural or cell-supporting hydrogel component, such as gelatin, gelatin methacrylate (GelMA), alginate, or combinations thereof, which forms a primary tissue matrix and provides a bio-permitted environment for cell encapsulation, migration, and tissue development. In a preferred embodiment, the matrix bioink is photocrosslinkable and may include additional agents such as photoinitiators and ionic crosslinking agents.

[0064] "Sacrificial bio-ink" refers to a printable hydrogel composition that is deposited to form a temporary structure in a bioprinted construct and subsequently removed under physiological or aqueous conditions, such as thermal liquefaction or enzymatic degradation. In some embodiments, the sacrificial bio-ink may further contain living cells, such as endothelial cells, which adhere to the exposed surface after the sacrificial material is removed.

[0065] "Perfusion channels" or "large channels" refer to space-defined cavities or conduits formed within a bioprinted tissue construct, sized for convective fluid transport and configured to allow the flow of nutrients, oxygen, or blood substitutes. These channels are typically created by removing sacrificial bio-ink and can be lined with endothelial cells to mimic the natural vascular system.

[0066] "Microporous matrix" refers to a hydrogel-based structure containing interconnected voids or pores, typically ranging in size from 5 μm to 100 μm. These micropores facilitate cell permeation, nutrient diffusion, and the self-assembly of microvascular networks, and can be formed by densely packed or blocked microgel particles.

[0067] "Fiber-like microgels" or "fibrous microgel particles" refer to suspensions of cross-linked hydrogel fragments or threads produced by splitting blocky hydrogels (e.g., through screen extrusion), having micrometer-scale diameters, and forming a clogged network upon packaging. These fibrous elements provide a microporous structure with enhanced rheological properties, including shear thinning and self-healing behavior.

[0068] "Self-healing support baths" refer to printable media that exhibit reversible thixotropic behavior—fluidizing under shear and rapidly re-solidifying after the shear stress is removed, such as fibrous microgel matrices. This property enables the media to support printed filaments during embedded or suspended bioprinting while maintaining structural integrity.

[0069] "Self-organized microvascular plexuses" refer to a network of microvessels or capillary-like structures formed spontaneously by embedded endothelial or vascular progenitor cells through a process of spontaneous morphogenesis. They are usually guided by matrix morphology, cell-cell interactions, and biochemical cues and do not require pre-patterned channel formation.

[0070] "Hierarchical porosity" refers to the presence of interconnected pore structures of various sizes within a tissue construct, typically comprising large, injectable macrochannels and smaller micropores or interstitial voids, which together facilitate convection and diffusion mass transport.

[0071] Other definitions of the selected terms used herein can be found throughout the detailed description of this invention and application. Unless otherwise defined, all other technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0072] In the following description, methods and systems for multicellular bioprinting of vascularized tissue constructs are illustrated as preferred examples. Those skilled in the art will recognize that modifications, including additions and / or substitutions, can be made without departing from the scope and spirit of the invention. Specific details may be omitted to avoid obscuring the invention; however, this document is written to enable those skilled in the art to practice the teachings herein without undue experimentation.

[0073] This invention provides a multicellular bioprinted tissue construct comprising an integrated hierarchical vascular network, along with a method for fabricating the tissue construct using dual bio-inks and a multi-nozzle 3D bioprinting system. This invention overcomes the limitations of conventional tissue engineering by enabling the scalable formation of perfusionable large vascular channels and bio-organized microvascular networks within a single construct. This invention provides a platform-level solution for engineering thick, functionally vascularized tissues suitable for regenerative medicine, disease modeling, and drug screening applications.

[0074] On one hand, this invention provides a method for fabricating vascularized tissue constructs through a multi-material, multi-cell bioprinting process. The method integrates two complementary vascularization mechanisms: (i) top-down bioprinting of perfusionable large vascular channels and (ii) bottom-up self-organization of capillary-like microvessels. The fabrication process involves using a programmable multi-nozzle 3D bioprinter to co-print at least two different bioinks (matrix bioink and sacrificial bioink) into a spatially defined 3D structure.

[0075] like Figure 1AAs shown, the method integrates two complementary mechanisms: (i) top-down bioprinting of perfusionable large vascular channels and (ii) bottom-up self-organization of capillary-like microvessels. The platform utilizes a programmable multi-nozzle bioprinter to achieve spatially controlled deposition of multiple bioinks, one used as a biodegradable matrix and the other as a sacrificial template. The biodegradable matrix bioink provides a multiporous, bio-permitted environment supporting cell migration, proliferation, and morphogenesis, while the sacrificial bioink forms spatially defined conduits that can be selectively removed post-printing to create open channels. Together, these components allow for the formation of multi-scale, continuous, and interconnected vascular structures within the construct.

[0076] In a preferred embodiment, the matrix bioink comprises a fibrous microgel consisting of gelatin, GelMA, alginate, and a suitable photoinitiator, forming a printable and crosslinkable hydrogel network. This fibrous matrix provides a porous, bio-permitted environment that facilitates nutrient transport, cell migration, and vascular morphogenesis. The sacrificial bioink comprises at least one hydrogel-forming component, such as hyaluronic acid or gelatin, and may optionally encapsulate endothelial cells. Upon deposition, the matrix bioink crosslinks via photopolymerization and / or ionic interactions, wherein calcium ions released from the sacrificial ink optionally promote in-situ crosslinking of the alginate component.

[0077] Sacrificial bio-ink is printed into a predetermined pattern and subsequently removed under mild aqueous or physiological conditions (such as thermal liquefaction or enzymatic degradation), thereby creating perfusionable macroscale channels within the construct. With cells loaded in the sacrificial ink, the released endothelial cells can adhere to the channel walls and promote in situ endothelialization. Simultaneously, the matrix and endothelial cells embedded in the matrix bio-ink self-assemble to form an integrated microvascular plexus guided by fibrous microstructures. This integrated manufacturing strategy enables the formation of multi-scale, continuous, and interconnected vascular systems within tissue constructs. By adapting the bio-ink composition, cell type, and printing geometry, the method can be adapted to a variety of tissue models, including organ-specific constructs for regenerative medicine, disease modeling, and drug discovery.

[0078] This invention further discloses three forms of bioprinting that utilize the properties of fibrous microgels for scalable tissue engineering:

[0079] Interleaved co-printing: Alternating deposition of matrix and sacrificial ink creates an interwoven network of channels, enabling patterned vascularization and spatially localized cell localization. In vivo data, compared to a pore-free control, confirmed successful integration and enhanced cell survival.

[0080] Embedded printing: By using fibrous microgels as a cell-permitted matrix, complex vascular patterns (e.g., liver lobule analogs) can be directly printed within the construct, overcoming the mass delivery limitations of bulk gels; and

[0081] Suspension printing: The state-of-the-art form uses fibrous microgels as a dynamic support bath. This enables simultaneous top-down printing of large blood vessels and bottom-up matrix-guided self-assembly of capillary plexuses, resulting in continuous, multi-scale vascular trees that mimic the structure of natural organs.

[0082] On the other hand, this invention provides an integrated multi-material bioprinting platform and a bioprinted tissue construct comprising an integrated hierarchical vascular network within a biocompatible, biodegradable matrix. The platform utilizes a programmable multi-nozzle 3D bioprinter and a dual bio-ink strategy to achieve spatially confined deposition of both the matrix and the sacrificial material. A key innovation of the system is the use of a fibrous microgel matrix bio-ink, exhibiting a microporous structure with interconnected voids that facilitate nutrient diffusion, cell infiltration, and capillary self-assembly. This fibrous microgel not only supports high cell viability and mass delivery but also provides morphological cues to guide embedded cells in self-organizing into capillary-like structures, thereby forming biologically relevant microvascular plexuses.

[0083] Simultaneously, sacrificial bio-ink is printed into predefined patterns and subsequently removed to create macroscale perfusion channels within the construct. These hollow catheters can be endothelialized to mimic natural vascular systems, resulting in a two-scale vascular network that closely replicates the structural and functional complexity of natural tissue. In a preferred embodiment, the matrix bio-ink is co-loaded with a population of endothelial cells (such as HUVECs and hMSCs), which synergistically enhances vessel budding, endothelial alignment, and lumen formation throughout the construct.

[0084] Figure 1B Examples of multicellular bioprinted tissue constructs incorporating hierarchical vascular networks are depicted. Perfusionable macrochannels are embedded in a printed matrix and can be seeded with endothelial cells, while mesenchymal cells co-encapsulated in the matrix undergo self-assembly to form microvessels. This integrated vascular system enhances nutrient diffusion and oxygen delivery throughout the construct, enabling the fabrication of thick, viable, and physiologically relevant engineered tissues.

[0085] The fibrous microgel exhibits a network of micropores composed of interconnected voids, mimicking the morphology of natural hepatic sinusoids and promoting bio-self-assembly. Its tunable mechanical properties and self-healing rheological properties make it suitable as both a tissue matrix and a support bath for complex embedded or suspension printing. In a preferred embodiment, this bio-ink can form a capillary network through encapsulated endothelial and stromal cells without requiring pre-patterned microchannels.

[0086] The methods and systems of this invention demonstrate the potential to generate large-scale vascularized tissue constructs with possible applications in tissue engineering and regenerative medicine. Further development may involve optimizing printing parameters, exploring alternative cell types and biomaterials, and conducting scalability studies for potential clinical applications.

[0087] Example

[0088] Example 1 - Materials and Methods

[0089] Multi-nozzle bioprinting process

[0090] In one embodiment, a multi-nozzle direct ink writing (DIW) bioprinting process is used to fabricate multimaterial and / or multicellular constructs. The system used is a GeSiM DIW bioprinter equipped with three independent pneumatic pressure-based dispensing modules, each digitally controlled to enable programmable deposition of different bioinks. Each bioink formulation is loaded into a separate sterile cartridge dispenser selected based on the rheological properties of the corresponding ink and coupled to nozzles of different inner diameters. Prior to bioprinting, the temperature control platform and dispensing modules are preset to the desired operating temperature (typically between 15°C and 25°C) and maintained under stable conditions for at least one hour to ensure thermal equilibrium.

[0091] Printing parameters were systematically optimized to ensure continuous and uniform filament extrusion. Specifically, the printing speed was controlled within the range of 6 mm / s to 8 mm / s, and the pneumatic extrusion pressure was adjusted between 30 kPa and 100 kPa, depending on the specific viscosity and shear thinning behavior of each bio-ink. In addition to pressure calibration, the volumetric flow rate was monitored and maintained between 0.05 mL / min and 0.2 mL / min, depending on the nozzle diameter and material properties, to ensure dimensional fidelity and reproducible deposition.

[0092] Immediately after the deposition process, the sample was exposed to a wavelength of 405 nm and an intensity of 25 mW / cm². 2 Visible blue light is applied for 60 seconds to induce rapid network polymerization of the photoreactive GelMA components, thereby photocrosslinking the printed construct. In some embodiments, an optional ionic crosslinking step is performed by immersing the crosslinked construct in a sterile aqueous solution of calcium chloride (CaCl2) at a concentration ranging from 0 wt% to 1 wt% for 1 to 3 minutes, thereby promoting further ionic crosslinking of the alginate components within the matrix. The construct is then thoroughly rinsed with phosphate-buffered saline (PBS).

[0093] The resulting constructs were then immersed in Duchenne Modified Eagle Medium (DMEM) and transferred to a humidified cell culture incubator maintained at 37°C and 5% CO2 for post-manufacturing incubation and cell recovery. All procedures, including bioink preparation, system calibration, bioprinting, photocrosslinking, and post-processing, were performed under aseptic conditions to ensure the sterility of the entire manufacturing workflow.

[0094] Cell-loaded bio-ink formulation

[0095] In one embodiment, a cell-loaded matrix hydrogel bioink is prepared under aseptic conditions. Specifically, 1 mL of sterile matrix hydrogel ink is aspirated into a sterile disposable syringe. Additionally, a concentrated cell suspension is prepared by resuspending the desired cell population in sterile culture medium supplemented with fibronectin at a final concentration of approximately 1 mg / mL, resulting in a 1 × 10⁻⁶ cell suspension. 6 Cells / mL to 1×10 7 Cell density in the range of cells / mL.

[0096] The hydrogel ink injector and the cell suspension injector are connected via a sterile dual-lumen mixing interface (such as a Y-connector or a two-way valve). The contents of the two injectors are mixed by gently and alternately actuating the plunger during push-pull movements, thereby ensuring uniform distribution of cells throughout the hydrogel matrix while minimizing bubble formation and mechanical shearing of the cells.

[0097] After mixing, the resulting homogeneous, cell-loaded hydrogel ink is transferred to a sterile dispensing cassette or syringe pre-loaded with a suitable printing needle (e.g., with an inner diameter selected based on the bioink viscosity and cell size). The formulation can then be immediately used in subsequent bioprinting processes involving DIW or extrusion-based deposition.

[0098] Cell staining and imaging

[0099] Cells are stained using a membrane-permeable fluorescent dye to enable subsequent visualization and tracking of cells within the printed construct. Specifically, CellTracker... TM Blue or CellTracker TM Invitrogen (deep red dye) TM Thermo Fisher Scientific uses this technology to pre-label cells before bioprinting.

[0100] In the absence of serum, the dye was diluted to a final working concentration of 1 μM to 10 μM in DMEM. The resulting staining solution was added to adherent or suspension cell cultures and incubated at 37°C for 30 minutes under standard culture conditions. After incubation, the cells were washed three times with fresh culture medium or PBS to remove excess dye. The stained cells were then collected by centrifugation, resuspended in sterile buffer or a bio-ink compatible solution, and subsequently used for encapsulation in bio-ink or direct printing.

[0101] Immunofluorescence staining

[0102] Immunofluorescence staining was performed to visualize the cytoskeleton structure and endothelial markers in the bioprinted constructs. All procedures were performed under sterile conditions unless otherwise specified.

[0103] The samples were first washed with PBS and then fixed in 3.7% paraformaldehyde for 10 minutes at room temperature. After fixation, the samples were permeabilized with a standard permeabilization solution (e.g., PBS containing 0.1% Triton X-100).

[0104] For F-actin staining, a fluorescent phalloidin conjugate (e.g., F-actin-green-488) was diluted 1:40 in PBS and applied to the sample in the dark at room temperature for 40 minutes. After incubation, the sample was thoroughly washed with PBS containing 0.1% Triton X-100 to remove any unbound dye. The sample was then counterstained with 10 μg / mL 4',6-diamidinyl-2-phenylindole (DAPI) for nuclear visualization, followed by another PBS wash.

[0105] For CD31 immunostaining, the fixed and permeated sample was first blocked with 3% bovine serum albumin (BSA) in PBS at room temperature for 1 hour to minimize nonspecific antibody binding. The sample was then incubated overnight at 4°C with primary anti-CD31 antibody (e.g., diluted 1:100 in blocking buffer). After incubation, the sample was thoroughly washed with PBS containing 0.1% Triton X-100. Then, a fluorescent dye (e.g., Alexa Fluor) was applied at a 1:500 dilution. TM A secondary antibody conjugated with 594 anti-mouse or anti-rabbit IgG was prepared and incubated in the dark at room temperature for 1 hour. Final DAPI counterstaining (10 μg / mL) was performed as described above, followed by multiple PBS washes to remove excess staining.

[0106] The stained samples are then mounted on a glass slide or maintained in an imaging-compatible culture dish for subsequent fluorescence or confocal microscopy examination, allowing visualization of cytoskeleton tissue and endothelial marker expression in the 3D bioprinted construct.

[0107] In vivo transplantation and analysis

[0108] In vivo transplantation studies were conducted to evaluate the biocompatibility and integration of the bioprinted hydrogel constructs in a mammalian model. Cylindrical constructs with an inner diameter of 5 mm and a height of 2 mm were constructed using a previously described cell-loaded matrix bio-ink. Prior to transplantation, the constructs were cultured in vitro for 3 days under standard conditions (37°C, 5% CO2) to promote early microvascular network formation.

[0109] As a control, cell-free hydrogel constructs with the same geometry and composition were prepared and cultured under the same conditions.

[0110] The transplant procedure was performed using 10-week-old athymic naked (Nu / Nu) immunocompromised mice (n=3). All animal handling and surgery were performed in accordance with the institution’s animal care and use guidelines. Mice were anesthetized with inhaled isoflurane, and a small surgical incision was made to expose the left lateral lobe of the liver. PBS was injected subcapsularly to gently separate the membrane from the underlying parenchyma, forming a subcapsular sac for graft placement.

[0111] The bioprinted construct was then carefully inserted into the resulting subcapsular space. The incision site was sutured, and the mouse was returned to a standard environment for routine monitoring.

[0112] Seven days post-transplantation, the animals were euthanized, and the liver lobe containing the implanted construct was removed. Tissue samples were fixed in formalin, embedded in paraffin, and stained with hematoxylin and eosin (H&E). Tissue sections were examined for morphological evaluation, including assessment of hydrogel integrity, host tissue response, and preliminary evidence of construct-liver integration at the subcapsular interface.

[0113] Histological analysis

[0114] Histological analysis was performed to assess the structural integrity of the explanted hydrogel constructs and their interaction with host tissues after in vivo transplantation. Extracted samples were first fixed in 10% neutral buffered formalin at room temperature for 2 hours, then dehydrated using a graded ethanol series, and subsequently embedded in paraffin using standard histological techniques.

[0115] The paraffin-embedded sample was cut into 10 μm thick sections using a rotary microtome. The sections were then mounted on glass microscope slides and subjected to a standard dewaxing and rehydration procedure, which included sequential immersion in xylene, ethanol of decreasing concentrations (100%, 95%, 70%), and distilled water.

[0116] For H&E staining, rehydrated tissue sections are incubated with hematoxylin for 3 to 5 minutes, rinsed under running tap water, and, if necessary, differentiated in 1% acidic ethanol to reduce background staining. The sections are then blued in tap water or an alkaline solution, followed by counterstaining with eosin for 1 to 2 minutes. After staining, slides are dehydrated sequentially in graded ethanol, washed in xylene, and fixed with permanent coverslips.

[0117] All histological procedures were performed using standardized protocols to ensure reproducibility. Stained sections were then examined using a bright-field microscope to analyze tissue morphology, construct retention, inflammatory response, and integration at the graft-host interface.

[0118] Immunohistochemical staining

[0119] Immunohistochemical (IHC) staining was performed on tissue sections to detect and locate specific cell types in the explanted bioprinted constructs. This analysis aimed to confirm the presence and spatial distribution of hMSCs and HUVECs in the transplanted tissue.

[0120] Paraffin-embedded tissue sections were prepared and subjected to standard dewaxing, rehydration, and antigen retrieval procedures. After blocking endogenous peroxidase activity and nonspecific binding, the sections were incubated with primary antibodies specific to the target markers: CD133 antibody for identifying hMSCs and CD31 antibody for detecting HUVECs.

[0121] After incubation with the primary antibody, the slides were treated with a horseradish peroxidase (HRP)-conjugated secondary antibody according to the manufacturer's recommended protocol. Signal development was achieved using 3,3'-diaminobenzidine (DAB) tetrahydrochloride as the chromogenic substrate. Colorimetric development was performed for approximately 3 minutes, allowing brown staining to be visible at the antigen expression sites.

[0122] All steps were performed according to the antibody manufacturer's instructions. The stained slides were counterstained with hematoxylin in appropriate locations and examined under a bright-field microscope to assess cell localization, persistence, and potential tissue integration.

[0123] Example 2 - Preparation of Bio-ink

[0124] In this example, three types of bio-inks were prepared for the fabrication of multiscale vascularized tissue constructs, the three types of bio-inks being: (1) matrix hydrogel bio-ink, (2) fibrous microgel bio-ink, and (3) sacrificial bio-ink.

[0125] Matrix hydrogel bio-ink:

[0126] A biodegradable matrix hydrogel bioink was prepared by dissolving 2.5 wt% GelMA, 1–5 wt% gelatin, and 0.2 wt% phenyl-2,4,6-trimethylbenzoyl lithium phosphine (LAP) in PBS. The components were heated to 50 °C until completely dissolved and then sterilized using a 0.22 μm membrane filter. Sterile alginate powder (0–2 wt%) was then added to the solution under aseptic conditions. The resulting mixture was homogenized using a planetary centrifuge at 2500 rpm for 5 minutes to ensure uniform distribution.

[0127] Fiber-like microgel bio-ink:

[0128] To prepare fibrous microgel bioink, a pregel solution was prepared containing 0-0.2 g alginate, 0.25 g gelatin, 0.25 g GelMA, and 0.02 g LAP dissolved in 10 mL PBS. The solution was sterilized, transferred to a dispensing cassette, and refrigerated for approximately 1 hour to induce partial gelation. Then, a gelling agent with a strength of 25 mW / cm² was used. 2 Blue light is used for photocrosslinking for 20 seconds to stabilize the network. The crosslinked hydrogel is extruded through a 20 μm nylon sieve to produce a fibrous microgel with interconnected micropores. When cell encapsulation is required, a dual-syringe connector is used to mix the fibrous microgel with a concentrated cell suspension to achieve uniform distribution.

[0129] Sacrificial bio-ink:

[0130] The sacrificial ink consists of 5-7.5 wt% gelatin dissolved in deionized water containing 0-1.0 wt% calcium chloride. The solution is maintained at 24-27°C to preserve printability. In some embodiments, the sacrificial bio-ink is cell-loaded. For this purpose, HUVEC is applied at approximately 1 × 10⁻⁶. 6 Suspended at a concentration of 1 cell / mL, and immediately mixed with sacrificial ink before printing.

[0131] These bio-inks were subsequently used in the fabrication of vascularized constructs exhibiting both perfusionable macrochannels and microcapillary networks.

[0132] Example 3 - Characterization of fibrous microgels and bio-inks

[0133] This example describes the structural, rheological, and degradation-related characterization of a fibrous microgel matrix bioink used to fabricate hierarchical vascularized tissue constructs.

[0134] To overcome the limited mass transport associated with conventional bulk hydrogels, fibrous microgel matrices were developed. Unlike solid bulk hydrogels with nanoporous networks, fibrous matrices consist of discrete microgel particles, forming a clogged, interconnected microporous structure. Figure 2A As shown, confocal micrographs confirm the presence of micron-sized pores distributed throughout the construct. These interconnected micropores significantly increase the available surface area and pore space, thereby enhancing permeability and cell infiltration, a key feature of engineered thick, metabolically active tissues.

[0135] The fibrous microgels also exhibited significant thixotropic behavior, characterized by shear thinning and self-healing properties. Rheological measurements were performed using a five-step alternating strain scheme. Figure 2B As shown, under alternating low (1%) and high (100%) strain conditions, the fibrous microgels undergo a reversible transition between solid (G′>G″) and fluid (G″>G′) states. During high strain, the storage modulus (G′) drops below the loss modulus (G″), indicating fluidization, and fully recovers upon returning to low strain, demonstrating rapid self-healing. In contrast, conventional bulk gels consistently maintain a solid-like response. This self-healing capability enables the fibrous matrix to act as a support bath during embedded and suspended bioprinting of complex structures.

[0136] To evaluate mass delivery capability, diffusion measurements were performed using 2000 kDa fluorescent dextran. Figure 2C The results show that the dye rapidly permeates through the fibrous microgel matrix, indicating high permeability. In contrast, in the bulk hydrogel, the dye remains confined to the surface, with no observable permeation. These results confirm that the microporous structure of the fibrous microgel significantly enhances solute transport, contributing to improved nutrient diffusion and waste removal in large tissue constructs.

[0137] The post-printing stability and degradation of the construct were also characterized. For example... Figure 3A As shown, sacrificial bioink is removed by immersion in PBS, leaving perfusion channels in the matrix. Quantitative gravimetric analysis ( Figure 3B This indicates that approximately 50% of the initial construct mass was retained after the removal of the sacrificial ink. After a 10-day incubation period, constructs with higher calcium ion concentrations exhibited increased structural integrity and slower degradation, such as... Figure 3C As demonstrated, these properties allow for gradual matrix renewal and potential replacement of natural extracellular matrix components during tissue maturation.

[0138] Example 4 - Adjustability of mechanical properties of bio-inks obtained through controlled photocrosslinking

[0139] This example demonstrates the ability to tune the mechanical stiffness of engineered hydrogel constructs formed from the disclosed bio-ink, thereby enabling the constructs to mimic the compressive modulus of natural soft tissues such as the liver.

[0140] This example demonstrates that the mechanical properties of hydrogel constructs formed from the disclosed bio-ink are tunable to mimic the stiffness of target natural tissues. The constructs were made from (i) a fibrous microgel matrix bio-ink (MF) and (ii) a matrix bulk hydrogel containing the same basic components but lacking the fibrous microstructure. After printing, the constructs were exposed to 25 mW / cm² water. 2 Photocrosslinking is performed under 405nm blue light for 30, 60, 120 or 240 seconds.

[0141] Unconfined compression tests were performed on the cross-linked constructs to determine the compressive modulus. Native mouse liver samples were tested under the same conditions as a physiological baseline. The compiled results are presented in… Figure 4 middle.

[0142] like Figure 4 As shown, both the MF and Bulk constructs exhibited a dose-dependent increase in compressive modulus with increasing exposure time. At matched exposure durations, the Bulk construct consistently showed a higher modulus than the MF construct, reflecting the microstructural differences between the bulk and fibrous matrices. Importantly, in one embodiment, the MF construct crosslinked for approximately 60 seconds produced a compressive modulus not significantly different from that of native mouse liver (shown as ns in Figure 2), while longer exposures (e.g., 120–240 seconds) produced progressively stiffer materials. The bulk structure showed a similar monotonic trend, reaching a modulus exceeding that of the MF at kPa levels across all exposure times.

[0143] These findings confirm that exposure duration provides a practical control knob for precisely adjusting construct mechanics. This adjustability allows the disclosed platform to be matched to tissue-specific stiffness (e.g., liver-like for MF-60 seconds) or intentionally deviated to a stiffer protocol when other goals require it, thereby broadening its applicability in regenerative medicine, disease modeling, and drug screening.

[0144] Example 5 - Fabrication of Dual Bio-Ink for Multi-Scale Vascularized Constructs

[0145] This chapter details three different but related printed structures (prototypes) that utilize the disclosed bio-inks to generate increasingly complex and functional vascularized tissues.

[0146] Example 5A

[0147] This example demonstrates the fabrication of vascularized tissue constructs using a dual-bio-ink printing method, where a structural matrix bio-ink and a removable sacrificial bio-ink are co-printed in an alternating pattern to create perfusion channels within a microporous support matrix. The fabrication process corresponds to... Figure 5A The illustrative steps shown and Figure 5B The experiment shown verifies this.

[0148] like Figure 5A As shown in (i), a multi-nozzle bioprinter is used to deposit alternating filaments of matrix bioink and sacrificial bioink on a substrate. The matrix bioink comprises a blend of GelMA, gelatin, and optionally alginate, forming a microporous and biocompatible support phase. The sacrificial bioink consists of hyaluronic acid and calcium ions, and in some embodiments includes embedded HUVECs. Figure 5A As shown in (ii), the construct undergoes in-situ crosslinking immediately after printing. Photocrosslinking of the GelMA component is performed using blue light irradiation (25 mW / cm²) for 30 seconds. Simultaneously, calcium ions from the sacrificial ink diffuse into the adjacent matrix bio-ink, promoting ionic crosslinking of the alginate chains. This dual crosslinking strategy stabilizes the construct while maintaining interconnected microporosity. Figure 5A As shown in (iii), the construct was then incubated at 37°C in an aqueous environment to dissolve the hyaluronic acid-based sacrificial phase. Removal of the sacrificial ink resulted in the formation of open, perfusionable large channels within the cross-linked matrix. Under cell-loaded conditions, endothelial cells derived from the sacrificial ink adhered to the inner surface of the formed channels, thereby achieving in situ endothelialization.

[0149] like Figure 5B As shown, the performance of this method was further validated experimentally. Calcium-free printed constructs ( Figure 5B (i) shows structural collapse, while using 1wt% Ca 2+ Printed builder ( Figure 5B (ii) Formation of a well-defined lattice structure. Using 250 μm (25G, Figure 5B (iv)) and 150μm (30G, Figure 5B The nozzles (v-viii) were used to create multiple grid and line patterns to evaluate resolution and material fidelity. The resulting constructs exhibited high pattern fidelity and stable cross-linked geometry, suitable for subsequent perfusion and cell culture.

[0150] In summary, this dual-bio-ink approach enables the fabrication of vascularized constructs comprising engineered perfusionable macrochannels and microporous cell-licensed matrices. This strategy facilitates spatially controlled vascular patterning and provides a solid foundation for hierarchical tissue engineering.

[0151] Example 5B

[0152] In one embodiment, a staggered co-printing strategy is used to fabricate three-dimensional vascularized tissue constructs, enabling the spatially defined deposition of multiple cell populations and sacrificial elements. For example... Figure 6A As shown, a multi-nozzle direct ink writing system is used to co-deposit at least one matrix bio-ink and a sacrificial bio-ink using alternating filament arrangements. The matrix bio-ink provides long-term structural support and cell encapsulation, while the sacrificial ink acts as a temporary placeholder for subsequent large channel formation.

[0153] like Figure 6B As shown, orthogonally arranged filaments of matrix and sacrificial ink are sequentially deposited layer by layer to construct a lattice-like three-dimensional structure. After printing, the matrix is ​​cross-linked using light radiation, and the sacrificial components are subsequently removed by thermal or enzymatic methods, resulting in interconnected, permeable macrochannels. This process leads to the formation of a porous structure with internal conduits suitable for fluid transport and intercellular delivery.

[0154] To assess the function of this structure in supporting tissue formation, a density of approximately 1 × 10⁻⁶ primary hepatocytes was used. 7 The matrix bio-ink was prepared using cells / mL and interleaved with cell-free sacrificial ink to create constructs. For example... Figure 7A As shown, the resulting construct exhibited robust structural integrity. After 48 hours of incubation, compaction of the matrix chains loaded with hepatocytes was observed, forming a dense, cord-like tissue region consistent with early tissue morphogenesis. Figure 7B ).

[0155] In another embodiment, a co-printing configuration using three inks is employed to deposit three different bio-inks, each containing fluorescently labeled human hepatocytes (LO2), endothelial cells (HUVEC), and immune cells (THP-1). Figure 7C As shown in (i)-(iii), the printed cell filaments maintain clear spatial separation within the lattice structure. Importantly, after 24 hours of incubation, HUVECs embedded in sacrificial ink were observed to adhere to the inner surface of adjacent matrix filaments, forming an endothelial-like lining along the large channel walls. Figure 7C (iv)). This phenomenon indicates in situ endothelialization induced by programmed release and adhesion.

[0156] Further experiments involved the co-printing of hMSCs in matrix ink and HUVECs in sacrificial ink, demonstrating the ability to engineer tissue constructs using regionally co-located cell populations. For example... Figure 7D As demonstrated by confocal imaging, hMSCs remain confined within the stromal scaffold, while HUVECs migrate and adhere to the stromal interface, establishing a spatial proximity that facilitates angiogenesis signal transduction and lumen formation.

[0157] To evaluate the biointegration potential of the engineered constructs, interleaved constructs loaded with cells were transplanted into the subcapsular liver region of immunodeficient mouse hosts. Constructs fabricated from bulk cell-free hydrogels were used as controls. Figures 8A-8B The printed constructs demonstrated maintained their anatomical positioning after implantation. Hematoxylin and eosin (H&E) staining of the explant tissues revealed significant differences in host response. In the control group, the hydrogel interface remained non-integrated and cell-free. Figure 8C (i)). In contrast, the printed constructs exhibited extensive host cell infiltration and a continuous interface with adjacent liver tissue (i). Figure 8C (ii)).

[0158] To confirm the persistence and phenotype of human cells, immunohistochemical staining was performed on explanted tissue samples. The human endothelial marker CD31 was detected along the tubular structures within the construct. Figure 8D (i)), while the mesenchymal stem cell marker CD133 was detected in the stromal region. Figure 8D (ii)) confirmed the in vivo survival and spatial organization of the implanted hMSCs and HUVECs.

[0159] These results collectively demonstrate that the staggered co-printing structures disclosed in this paper support: (i) precise deposition of multiple bio-inks and cell types, (ii) programmable formation of perfusion channels, (iii) in situ endothelialization without secondary seeding, and (iv) successful in vivo integration and persistence of human tissue components. This strategy enables the fabrication of hierarchical, functional tissue constructs with potential applications in regenerative medicine, disease modeling, and vascularized tissue replacement.

[0160] These in vivo results highlight the biological relevance of the interleaved co-printed structures. The porous constructs allow for extensive infiltration of host blood cells and human CD31. + and CD133 + The presence of the biomarkers confirmed the successful transplantation and survival of endothelial and mesenchymal stromal cell populations. This supports the utility of the disclosed structures for future clinical translation and long-term regenerative applications.

[0161] Example 5C

[0162] In one embodiment, vascularized tissue constructs are fabricated via embedded bioprinted structures, wherein sacrificial bio-ink is deposited directly into a fibrous microgel (MF) matrix, which simultaneously serves as a biofunctional tissue scaffold and a shear-thinning support medium. This approach is configured to overcome the limitations associated with conventional bulk hydrogels, particularly regarding their nanoporous structure and constrained mass delivery properties.

[0163] like Figure 9A The embedded printed structure, schematically illustrated, is inspired by the hierarchical vascular design of liver lobules, which consist of a network of sinusoidal spaces radiating towards the central vein. To generalize this hexagonal microanatomy, a three-layer lattice printing scheme was implemented, such as... Figure 9B Detailed explanation.

[0164] The process includes the following steps:

[0165] (i) Formation of the support matrix: The previously crosslinkable and microporous fibrous microgel bio-ink (bio-ink A) is dispensed into a printing reservoir or mold to define a receiving tissue matrix and a support bath;

[0166] (ii) Embedded sacrificial patterning: Sacrificial bio-ink (Bio-ink B) optionally loaded with cells is extruded into the MF matrix to form a vascular template. Printing follows a rotating pattern in which each of three consecutive layers is deposited at a 60-degree angle offset, thereby producing a repeating hexagonal geometry;

[0167] (iii) Crosslinking and Stabilization: After sacrificial ink deposition, the entire construct is stabilized through photocrosslinking and / or iontophoresis, based on the specific functional groups of the MF matrix; and

[0168] (iv) Removal of sacrificial components: The sacrificial ink is selectively removed under aqueous or thermal conditions to create a perfusionable large-channel network embedded in the MF-based tissue matrix.

[0169] like Figure 9C As shown, the resulting construct comprises a hierarchical, multi-scale porous structure consisting of: (i) an engineered macroscale vascular conduit formed by sacrificial ink removal, which defines a triangular tissue region arranged in a biomimetic lobule configuration; and (ii) the inherent microscale interstitial porosity of the material provided by the fibrous microgel matrix itself, which enhances nutrient diffusion and cell remodeling within each lobule region.

[0170] Notably, each triangular tissue region is defined by converging perfusion channels and is entirely constructed of MF material, thereby providing continuous, interconnected micropores throughout the construct. This structural feature offers dual delivery advantages: macroscale perfusion via vascular catheters and localized mass transfer within tissue regions.

[0171] Compared to the staggered co-printing strategy described in Example 5B, the structure of the present invention enables higher structural accuracy, improved perfusion uniformity, and closer anatomical simulation of tissues with lobular or nodular morphologies. Specifically, angular control of the sacrificial lattice orientation allows for the generation of isotropic or anisotropic vascular regions, enabling applications to a wide range of organ systems, including but not limited to the liver, kidneys, and pancreas.

[0172] Example 5D

[0173] In one embodiment, multi-scale vascularized tissue constructs are fabricated by suspension bioprinting, wherein sacrificial bio-ink is deposited in a fibrous microgel (MF) support bath, which simultaneously serves as (i) a self-healing, shear-thinning deposition medium and (ii) a biocompatible microporous tissue matrix.

[0174] like Figures 10A-10B The manufacturing process shown includes the following steps:

[0175] (i) Fill a custom-designed chamber made of polydimethylsiloxane (PDMS) with MF bio-ink to form a physically stable support matrix;

[0176] (ii) Sacrificial bio-ink is extruded into an MF bath along a pre-programmed three-dimensional trajectory configured to form a closed-loop large vessel circuit consisting of two terminal perfusion ports and four interconnected hexagonal units simulating the hepatic lobular vascular system.

[0177] (iii) After sacrificial ink deposition, the MF matrix is ​​crosslinked via photopolymerization to stabilize the construct; and

[0178] (iv) Remove sacrificial bio-ink under aqueous or thermal conditions to create open, large, infusible channels, in which infusion needles are inserted at the inlet / outlet to allow external fluid to enter.

[0179] This manufacturing process enables the formation of a hierarchical vascular system comprising: (i) engineered large channels capable of convective perfusion, and (ii) micropore-based regions supporting local diffusion and intercellular transport. Figure 10B As shown, this structure establishes a multi-scale cyclic network with integrated functions within the construct.

[0180] To evaluate the endothelialization capability of engineered large channels, sacrificial gelatin-based bio-ink containing human umbilical vein endothelial cells (HUVECs) was printed into a cell-free MF bath. When the sacrificial ink liquefied at physiological temperature (37°C), the HUVECs were released into the lumen of the newly formed channels. Figure 11A ).

[0181] Fluorescence microscopy on day 1 confirmed that HUVECs adhered uniformly along the interior of the channels. Figure 11B (i)). By day 7, HUVECs exhibited proliferation and directed budding into the surrounding MF matrix, forming radially arranged angiogenic structures. Figure 11B (ii)). These results indicate that suspension-printed channels not only support in situ cell deposition but also promote functional endothelialization and cell interface integration.

[0182] In a separate embodiment, the MF matrix was preloaded with a co-culture of HUVECs and human mesenchymal stem cells (hMSCs) to assess its ability to support spontaneous microvascular tissue.

[0183] like Figure 12A As shown, by day 10 of in vitro culture, endothelial cells formed an interconnected network of capillary plexuses throughout the MF scaffold. By day 14, further network maturation was observed, including the formation of hollow endothelial lumens, thus confirming the establishment of physiologically relevant capillary-like microvessels. Figure 12B In summary, these results validate the suspension printing method as a powerful platform for fabricating multi-scale vascularized tissues, successfully combining engineered perfusionable vascular channels with self-assembled microcapillaries.

[0184] Example 6 - Structure-guided cell patterning using fibrous microgels

[0185] Example 6A

[0186] This example demonstrates the application of the disclosed dual-bio-ink bioprinting platform in the fabrication of engineered tissue constructs comprising one or more cell types distributed within a spatially defined structure. Specifically, the method enables high-density cell encapsulation, void channel formation, and intercellular interactions within the printed matrix and sacrificial regions.

[0187] In one embodiment, the cell-loaded matrix ink is compatible with cell densities ranging from 1.25 million to 12.5 million cells per mL, with cell viability observed at higher densities up to 94%. Figure 13A Single-cell tissue constructs were created using dual bio-inks with cross-printing patterns. Figure 7A After removing the sacrificial ink, void channels and cell diffusion from the matrix ink were observed. Figure 13B (i) and (ii)). After 48 hours of culture, the cells in the matrix formed a dense structure. Figure 13B (iii)). These features may facilitate nutrient exchange and oxygen diffusion within engineered tissues.

[0188] In another embodiment, multicellular constructs are prepared by co-printing at least one matrix bioink (M) and two different sacrificial bioinks, each loaded with a different cell population. An interleaved deposition pattern is employed. Figure 13C After removing the sacrificial ink and incubating for 24 hours, cells from the sacrificial ink aligned along the surface of the matrix filaments, forming luminal structures with adherent cells, marked as channels ( Figure 13D This technology enables spatial control of cell type localization and interaction dynamics, which is crucial for modeling complex tissue microenvironments.

[0189] Example 6B

[0190] In one embodiment, the structural guidance capability of fibrous microgels is evaluated in the absence of predefined large vascular channels or patterned cues to determine their ability to replicate natural sinusoidal microstructures.

[0191] Histological analysis of the rat liver revealed a densely branched network of sinusoids defined by narrow spaces between hepatocyte plates. Figure 14A It is speculated that the interconnected microporous structure of the fibrous microgel, containing hydrogel filaments in the 20-50 μm diameter range, can serve as a physical scaffold to guide vascular cells to self-organize into a biomimetic sinus-like morphology.

[0192] To test this hypothesis, a co-culture of human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs) was uniformly mixed into a fibrous microgel bio-ink to form a cell-loaded matrix without any printed vascular patterns. The construct was cultured for 14 days under standard in vitro conditions.

[0193] Confocal microscopy revealed that the embedded cells elongated along the microgel filaments and self-organized into a continuous network of capillary reticles. The cellular structure followed the morphological guidance of a fibrous framework, encased around the microgel chains, and formed branching structures throughout the matrix. Figure 14B-14C ).

[0194] The resulting morphological reproducibility reproduced key features of the hepatic sinusoids, including luminalized interstitial spaces and well-organized capillary branches. These findings demonstrate that the fibrous microgel not only serves as a permitting scaffold but also acts as an active morphological indicator of the environment. Such biomimetic structures have significant value for tissue-specific engineering of the liver and other organs rich in sinusoids.

[0195] Example 7 - Bioengineered capillary networks co-cultured in microfiber hydrogels

[0196] This bioprinting method involves the use of specialized matrix bioinks. Microfibril matrix cell inks for vascular network formation were explored. Endothelial cells were encapsulated within the microfibril structure bioink. Figures 15A-15D The cells in the microfiber bioink maintained an elongated, stretched morphology compared to their round shape in the bulk gel.

[0197] A 1:1 ratio of HUVECs and hMSCs co-culture system was used. Observations were made during a 14-day culture period. Figure 16 and 17 This demonstrates cell diffusion and morphology under co-culture conditions. By day 7, co-cultured endothelial cells began to form bud structures. This continued with prolonged culture, resulting in an interconnected microcapillary network by day 10. By day 14, the presence of hollow endothelial lumens confirmed the successful formation of a functional microvascular network. These results validate that the microfibrillary matrix, as a bioactive scaffold, can guide microvascular morphogenesis through intrinsic cellular mechanisms.

[0198] Example - In-situ development of large blood vessel structures using sacrificial bio-ink loaded with HUVECs

[0199] This example illustrates a dual-chamber vascularization method in which large perfusion channels are generated using time-delayed, sacrificial bio-ink loaded with endothelial cells.

[0200] This method integrates microfiber matrix ink with time-delayed sacrificial bio-ink. Figure 6A The platform allows for the generation of vascularized tissue constructs, such as those demonstrated by printing in a 4-well cell culture chamber. Figure 18A After printing, the structure undergoes a second photopolymerization. Figure 18B ).

[0201] To elucidate the vascularization process, cell-free microfiber ink and HUVEC-encapsulated sacrificial ink were used. 7.5% gelatin was used as the sacrificial material. Initially, as the gelatin dissolved, endothelial cells formed clusters and adhered to the surface of the matrix hydrogel. Figure 18C By day 7, these cells invade the surrounding matrix tissue and develop buds, elongating to form vascular structures. Figure 18D-18E ).

[0202] This dual bio-ink method allows for the formation of two distinct vascular components within the tissue construct: a microfibril matrix supports capillary network formation, while the sacrificial gelatin ink creates larger channels lined with endothelial cells.

[0203] The foregoing description of the invention has been provided for purposes of illustration and description. The foregoing description is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations will be apparent to those skilled in the art.

[0204] The embodiments were chosen and described in order to best explain the principles of the invention and its practical application, thereby enabling others skilled in the art to understand the invention with respect to the various embodiments and with various modifications suitable for the particular purpose considered.

[0205] Industrial applicability:

[0206] This invention has wide industrial applications, especially in the fields of tissue engineering, regenerative medicine and basic research.

[0207] Its main functions include creating complex three-dimensional vascularized tissue constructs, precisely controlling cell distribution within engineered tissues, and forming multi-scale vascular networks ranging from capillaries to larger vessels. This invention supports high cell viability in bioprinted constructs and promotes efficient nutrient exchange and oxygen diffusion within these tissues. In tissue engineering, it can be used to develop more complex and functional tissue constructs and create tissue models for various organ systems. In regenerative medicine, this invention has the potential to develop implantable tissue constructs. It is also useful for basic research, providing insights into vascularization processes, cell-cell and cell-matrix interactions in a three-dimensional environment. Furthermore, its application in multicellular tissue engineering allows for the integration of multiple cell types in precise spatial arrangements, replicating complex tissue microenvironments.

Claims

1. A method for multi-cell bioprinting of vascularized tissue constructs, characterized in that, The method includes: Prepare a biodegradable matrix bio-ink containing gelatin methacrylate (GelMA) and gelatin; Preparation of sacrificial bio-inks containing hydrogel-forming components and calcium agents; The matrix bio-ink and the sacrificial bio-ink are loaded into separate cartridges of a multi-nozzle 3D bioprinter; The matrix bio-ink and the sacrificial bio-ink are co-printed in alternating or embedded patterns to form a three-dimensional tissue construct; The matrix bio-ink is crosslinked and the sacrificial bio-ink is removed to create perfusion channels in the three-dimensional tissue construct; as well as The vascularized tissue construct is formed by integrating a biodegradable matrix and a perfusionable channel.

2. The method according to claim 1, wherein the matrix bioink further comprises lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as a photoinitiator.

3. The method of claim 1, wherein the matrix bio-ink comprises microporous fibrous microgel particles formed by splitting cross-linked block hydrogel, and the fibrous microgel particles are prepared by extruding the cross-linked hydrogel through a 20 μm sieve.

4. The method of claim 1, wherein the sacrificial bio-ink comprises a density of 5 × 10⁻⁶. 5 10 cells / mL to 2×10 6 Endothelial cells in the range of cells / mL, and said endothelial cells adhere to the surface of the inner channel after the sacrificial bio-ink is removed.

5. The method of claim 1, wherein the three-dimensional tissue construct comprises a hexagonal large-channel geometry that mimics the natural liver lobule.

6. The method of claim 1, wherein the sacrificial bio-ink is directly printed into a self-healing support bath, the self-healing support bath comprising a microporous matrix blocked by fibrous microgels. The self-healing support bath self-heals around the printed filaments prior to crosslinking and contains a first cell population, enabling the three-dimensional tissue construct to exhibit a hierarchical porosity defined by the printed large channels and inherent microporosity, and supporting capillary self-assembly within the matrix.

7. The method of claim 6, wherein the printed large channel defines a injectable circuit, the injectable circuit comprising at least one inlet and at least one outlet.

8. A vascularized tissue construct for implantation into a host organ, characterized in that, The vascularized tissue construct comprises: A microporous matrix with interconnected micropores formed from a bio-ink composition; An infusible network extending through large channels into the microporous matrix; as well as One or more populations of living cells encapsulated within the microporous matrix, wherein the vascularized tissue construct exhibits hierarchical porosity defined by the macrochannels and the interconnected micropores of the microporous matrix; and The vascularized tissue construct comprises a self-organized microvascular plexus formed within the microporous matrix.

9. The vascularized tissue construct of claim 8, wherein the bio-ink composition comprises a suspension of cross-linked hydrogel microparticles including GelMA and gelatin, wherein the suspension of cross-linked hydrogel microparticles forms a blockage microporous matrix having interconnected pores in the range of 20 μm to 50 μm. Furthermore, the microporous matrix described therein exhibits shear thinning and self-healing thixotropic properties.

10. The vascularized tissue construct of claim 8, wherein the interconnected micropores are configured to guide vascular cells to form sinusoidal capillary structures.

11. The vascularized tissue construct of claim 8, wherein the inner surface of the large channel is lined with endothelial cells derived from the sacrificial bioink.

12. The vascularized tissue construct of claim 8, wherein the encapsulated cells comprise a co-culture of human mesenchymal stem cells and endothelial cells, and wherein the endothelial cells, guided by the morphological features of the matrix, self-organize into branched capillary plexuses along the interstitial spaces of the microporous matrix.

13. The vascularized tissue construct of claim 8, wherein the perfusionable network of the large channels comprises at least one inlet and at least one outlet, the at least one inlet and the at least one outlet being configured to allow bidirectional perfusion through the vascularized tissue construct.

14. The vascularized tissue construct of claim 8, wherein the microporous matrix simulates the microstructure of the hepatic sinusoidal space.

15. A system for bioprinting vascularized tissue constructs, characterized in that, The system includes: A programmable multi-nozzle 3D bioprinter with at least two independently controlled printheads, wherein at least one temperature control box dispenser is configured to dispense gelatin-based sacrificial bio-ink at 20-30°C; One or more temperature-controlled cartridge dispensers for bio-inks; A photocrosslinking device, the photocrosslinking device being configured to deliver blue light; and A chamber for accommodating a self-healing support bath during embedded or levitation printing.

16. The system of claim 15, wherein the system further comprises a dual-syringe Y-connector for mixing a cell suspension into a matrix bio-ink under aseptic conditions.

17. The system of claim 15, wherein the system further comprises nozzles of different sizes for generating vascular structures with different channel diameters.

18. The system of claim 15, wherein at least one independently controlled printhead is pneumatically pressure driven and digitally controlled to regulate extrusion.

19. The system of claim 15, wherein the system further comprises a microfiber bioink preparation apparatus, the microfiber bioink preparation apparatus comprising a nylon screen with a pore size of approximately 20.0 μm and a planetary centrifugal mixer.

20. The system of claim 15, wherein the system further comprises a PDMS chamber configured to contain the self-healing support bath during levitation printing.

Images