Methods for embedded 3D printing of functional constructs for biological, robotic or other applications

Abstract

A method of fabricating a functional construct for biological, robotic or other applications includes printing core-shell filaments into a support matrix, where each core-shell filament has an elongated core surrounded by a shell, and interconnecting the core-shell filaments to form branching and merging structures. Accordingly, an interconnected three-dimensional network of the core-shell filaments is formed within the support matrix. A tissue construct or other functional construct may be produced by this method.

Description

METHODS FOR EMBEDDED 3D PRINTING OF FUNCTIONAL CONSTRUCTSFOR BIOLOGICAL, ROBOTIC OR OTHER APPLICATIONSINVENTORS:Paul Philip StankeySebastien G. M. Uzel Daniel S. Reynolds Jennifer A. LewisRELATED APPLICATION

[0001] The present patent document claims the benefit of priority under 35 U.S.C. 119(e) to U.S. Provisional Patent Application No. 63 / 539,871, which was filed on September 22, 2023, and is hereby incorporated by reference in its entirety.FEDERALLY FUNDED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under CA214369 awarded by National Institutes of Health (NIH) and under N00014-21- 1-2958 awarded by U.S. Office of Naval Research (NAVY / ONR). The government has certain rights in this invention.TECHNICAL FIELD

[0003] The present disclosure relates generally to additive manufacturing and more particularly to three-dimensional (3D) printing of functional constructs for various applications.BACKGROUND

[0004] Biomanufacturing organ- specific human tissues replete with a biomimetic vascular network remains a formidable challenge. Native blood vessels are composed of concentrically arranged layers, in which the inner most layer (intima) is formed by a confluent endothelium that regulates barrier function. The endothelium is supported by smooth muscle cells (SMCs) which reside in the medial layer and improve vessel robustness. Without the ability to embedimmediately addressable and perfusable vasculature, engineered human tissues may not remain viable over the time required to provide therapeutic benefit. Recent advances in extrusion, embedded, and light-based bioprinting have begun to address this critical need. Yet no method currently allows the free-form patterning of hierarchical, branching vasculature comprising SMC-laden shells that surround endothelialized lumens in acellular or densely cellular tissue matrices.BRIEF DESCRIPTION OF THE DRAWINGS

[0005] The embodiments may be better understood with reference to the following drawing(s) and description. The components in the figures are not necessarily to scale. Moreover, in the figures, like-referenced numerals designate corresponding parts throughout the different views.

[0006] FIGS. 1A and IB illustrate three-dimensional (3D) printing of core-shell filaments into a support matrix, including the formation of branching and merging structures.

[0007] FIG. 2 shows an exemplary interconnected 3D network of the core-shell filaments that includes numerous branching and merging structures.

[0008] FIG. 3A shows a cross-sectional view of a printhead comprising a core-shell nozzle.

[0009] FIG. 3B shows details from the extended core tip of the core-shell nozzle from FIG. 3A.

[0010] FIGS. 4A-4D illustrate approaches to forming branching and merging (or reconnection) structures.

[0011] FIGS. 5A-5D show core-shell filaments composed of exemplary stiff, matched, and soft shell inks printed vertically within a granular alginate support matrix; scale bars are 250 pm.

[0012] FIGS. 6A-6D show core-shell filaments composed of the exemplary stiff, matched, and soft shell inks printed horizontally within a granular alginate support matrix; scale bars are 1 mm.

[0013] FIGS. 7A and 7B show rheological characterization of the exemplary core ink, shell inks (stiff, matched, and soft), and support matrix referred to above.

[0014] FIG. 8 shows an optical image of core-shell filaments formed into an interconnected 3D network (longitudinally sectioned view) in an acellular matrix comprising granular alginate microparticles, in which bifurcating channels follow Murray’s law; scale bar is 10 mm.

[0015] FIGS. 9A-9D show optical images (cross-sectional view) of the printed core-shell filaments for each order of the printed network shown in FIG. 8; scale bars are 1 mm.

[0016] FIG. 10 is a plot of total and core diameters for filaments printed at different speeds at a constant volumetric flow rate.

[0017] FIG. 11A and 11B show a 3D biomimetic vascular network formed from printed core-shell filaments before (FIG. 11A) and after (FIG. 11B) removal of the core ink and perfusion of the open vessels; scale bars are 10 mm.

[0018] FIG. 12 is a schematic representation of an exemplary core-shell printed filament in a granular collagen matrix prior to lumen formation via sacrificial ink removal.

[0019] FIG. 13 shows printed smooth muscle cell (SMC) viability compared to an as-cast control over seven-day period of vessel perfusion.

[0020] FIG. 14A shows a confocal image (longitudinal cross-section) of a branched, endothelialized vessel network produced by an implementation of the core-shell printing method (coaxial embedded printing or co-EMB3DP); scale bar is 1 mm.

[0021] FIG. 14B shows a higher magnification, confocal image of confluent endothelium lining the printed and perfused branching vessels of the endothelialized vessel network shown in FIG. 14A; scale bar is 50 pm.

[0022] FIG. 15 shows results from a permeability assay of bare and endothelialized vessels following one week of culture.

[0023] FIG. 16A shows a live / dead assay of vascularized cardiac tissue produced by an implementation of the core-shell printing method (coaxial sacrificial writing into functional tissues or co-SWIFT); white lines denote the edges of the vessel wall, and the scale bar is 500 pm.

[0024] FIG. 16B shows a cross-section of endothelialized cardiac tissue produced via coSWIFT following five days of culture. Scale bar is 100 pm.

[0025] FIG. 17A shows beat amplitude of cardiac co-SWIFT tissues over time.

[0026] FIG. 17B shows beat amplitude traces of cardiac co-SWIFT tissues following drug treatment.

[0027] FIG. 17C shows quantification of spontaneous beating rate of cardiac co-SWIFT tissues following drug treatment.DETAILED DESCRIPTION

[0028] Described herein is a core-shell printing technique to manufacture hierarchically branching and merging structures, which may extend in any or all directions, embedded into a support matrix. The three-dimensional (3D) printing method may utilize a printhead with a nozzle having an inner wall that extends beyond the outer wall to facilitate puncturing and interconnecting printed core-shell filaments, enabling the formation of three-dimensional (3D) vascular networks in various support matrices.

[0029] Applications for the method may include the creation of blood vessels to sustain therapeutically relevant tissue engineered constructs. For example, the method may be used to print core-shell filaments with a shell including protein (e.g., collagen and / or fibronectin) and cells (e.g., smooth muscle cells) into highly cell-dense granular tissue; upon removal of the sacrificial core of the core-shell filaments, perfusable blood vessels may be formed. In another example, the method may be applied to print core- shell filaments into granular protein materials either with or without cells for drug screening or disease modeling. Again, removal of the sacrificial core may allow for the formation of perfusable blood vessels. The printing method may also or alternatively be employed in non-biological applications where 3D networks may be advantageous, such as robotics.

[0030] Referring to FIGS. 1A and IB, the method of fabricating a functional construct 100 for biological, robotic or other applications includes printing core-shell filaments 102 into a support matrix 104, where each core-shell filament 102 comprises an elongated core 106 surrounded by a shell 108. The core- shell filaments 102 are interconnected to form branching and merging structures 110, such that an interconnected three-dimensional network 112 of the core-shell filaments 102 can be formed within the support matrix 104, as illustrated in FIG. 2. Each branching and merging structure 110 includes a junction 114 of two or more core-shell filaments 102, where, at each junction 114, the elongated cores 106 of the two or more coreshell filaments 102 are connected, and the shells 108 of the two or more core-shell filaments 102 are connected. The branching and merging structures 110 may have a geometry that is biomimetic, derived from a patient scan, or otherwise conceived. Upon removal of the elongated cores 106 (if desired), an interconnected three-dimensional network 112 of open vessels 102a may be formed.

[0031] As illustrated in FIG. 3A, printing the core-shell filaments 102 may entail, for each core- shell filament 102, flowing a core ink 116 and a shell ink 118 through a core- shell nozzle120, which has a core channel 122 surrounded by a shell channel 124 and is moving relative to the support matrix 104. The core-shell nozzle 120 is positioned at a downstream end of the printhead 140. The movement of the core-shell nozzle 120 relative to the support matrix 102 may occur in x-, y-, and / or z-directions, and thus the printing may be referred to as omnidirectional printing. The core ink 116 is flowed through the core channel 122 and the shell ink 118 is flowed through the shell channel 124. The core-shell nozzle 120 further includes an inner wall 126 surrounded by an outer wall 128. The inner wall 126 is spaced apart from the outer wall 128 by the shell channel 124, and the inner wall 126 surrounds the core channel 122. As can be seen in FIG. 3B, the tubular inner wall 126 extends longitudinally beyond the tubular outer wall 128 at an outlet end 130 of the core- shell nozzle 120, thereby defining an extended core tip 132.

[0032] As shown in FIGS. 4A-4D, interconnecting the core-shell filaments 102 may comprise puncturing shells 108 of the core-shell filaments 102 with the extended core tip 132. As illustrated, printing (or motion of the nozzle 120 relative to the support matrix 104) may be paused prior to puncturing and then resumed after puncturing. To create a branching structure, the extended core tip 132 may be used to puncture the shell 108 of a printed core-shell filament 102, e.g., at a midpoint or other location between endpoints of the printed filament as shown in FIG. 4A. The core and shell inks 116,118 may then be concurrently extruded to produce a junction 114 of the parent and daughter filaments 102, as shown in FIG. 4B. Printed filaments 102 may be reconnected in various ways. The extended core tip 132 may once again be used to puncture the shell of a printed filament 102. This may occur as the inks 116,118 are being extruded, as indicated in FIG. 4C, or while printing is halted. A junction may also or alternatively be formed at the free end of the printed filament 102, as indicated in FIG. 4D. These interconnections may be performed sequentially to build increasingly complex branching vascular networks.

[0033] It is preferred that the extended core tip 132 has a length comparable to or greater than a shell thickness. In some examples, the extended core tip 132 has a length of least about 10 microns, at least about 50 microns, at least about 100 microns, at least about 200 microns, or at least about 500 microns, and / or as large as about 1 cm, or as large as about 1 mm. Consistent with this, each of the core-shell filaments 102 may have a shell thickness in a range from about 10 microns to 1 cm. Also or alternatively, each of the core- shell filaments may have a core diameter in a range from about 10 microns to about 1 cm. The term “core diameter” maybe used although it is recognized that the cross-sectional shape of the core-shell filaments 102 may not be circular. Accordingly, the “core diameter” may be alternatively referred to as a “core width” or “core lateral dimension.” The inner and / or outer walls 126,128 of the nozzle 110, and thus the core-shell filaments 102, may have a profile or transverse cross-section which is circular, oval, elliptical, triangular, diamond- shaped, square, pentagonal, hexagonal, octagonal, star-shaped, irregular, or another polygonal shape. The inner and / or outer walls 126,128 may have different profiles or the same profile. The profiles or transverse crosssections of the core- shell nozzles 120 shown in FIGS. 3 A and 3B are both circular in this example. More specifically, the inner and outer walls 126,128 and the core and shell channels 122,124 shown in FIGS. 3 A and 3B are concentric.

[0034] Each of the core channel 122 and the shell channel 124 may be configured with a switching mechanism such as a solenoid valve or a rotary valve to control flow through the respective channel 122,124. Accordingly, the flow rate of the core ink 116 into the core channel 122 may be the same as or different from the flow rate of the shell ink 118 into the shell channel 124. A ratio of core diameter to shell thickness of the printed core-shell filaments 102 may be influenced or determined by the relative flow rates. For example, by increasing the flow rate of the core ink 116 relative to the flow rate of the shell ink 118, the ratio of core diameter to shell thickness may be increased. Alternatively, by decreasing the flow rate of the core ink 116 relative to the flow rate of the shell ink 118, the ratio of core diameter to shell thickness may be decreased. The ratio of core diameter to shell thickness may be varied during printing by altering the relative flow rates as the nozzle 102 moves relative to the support matrix 120. Alternatively, the flow rate of the core ink 116 relative to the shell ink 118 may remain constant during printing. Typically, a flow rate of the core ink 116 through the nozzle 120 ranges from greater than 0 to 10 cl / s, and a flow rate of the shell ink 118 through the nozzle 120 may also range from greater than 0 to 10 cl / s.

[0035] Another mechanism for tuning the filament / core diameter is by controlling print speed, that is, how fast the nozzle 120 is translating relative to the support matrix 104. As discussed below, filaments 102 of smaller diameters may be printed at higher print speeds. This control over print speed and / or ink flow rate may be particularly important when printing hierarchical branching and merging structures 110.

[0036] As shown in FIGS. 3 and IB, the core channel 116 and the shell channel 118 of the nozzle 120 may be coaxial, and the elongated core 106 and the shell 108 of the printed core-shell filament 102 may be coaxial. In other examples, the core channel 116 and the shell channel 118 may not be coaxial, and the elongated core 106 and the shell 108 may not be coaxial. For example, the core-shell nozzle 120 may include two or more of the core channels 116 surrounded by a single shell channel 118. Accordingly, the core-shell filaments 102 may in some examples include two or more of the elongated cores 106 surrounded by a single shell 108.

[0037] The shell ink 118 may have a stiffness higher than that of the support matrix 104 to ensure the stability and mechanical integrity of the printed core-shell filaments 102. As discussed in the examples below, it is preferred that a ratio of shear yield stress (ry) of the core ink to the shear yield stress (Ty) of the support matrix, which may be referred to as the corematrix Tyratio, is close to unity, e.g., 1 + / - 0.3. Also, it may be beneficial that a ratio of shear yield stress (ry) of the shell ink to the shear yield stress (ry) of the support matrix 104, which may be referred to as the shell-matrix ryratio, is at least 5, or at least 10, e.g., 10 + / - 3, for coaxial embedded printing. The core ink 116 may comprise a fugitive or sacrificial ink, that is, an ink that is ultimately removed. Accordingly, the method may further comprise, after forming the interconnected three-dimensional network 112 of the core-shell filaments 102, removing the fugitive or sacrificial ink (e.g., by heating and thereby liquefying the ink for extraction), such that the core-shell filaments 102 are replaced with open vessels 102a, as indicated in FIG. 2. That is, only the shell 108 remains after removal of the core 106. In some examples, printing may take place at a reduced temperature (e.g., below room temperature (18-22°C) and / or below a curing or gelation temperature of the support matrix 104) and thus may entail cooling the printhead / nozzle 140,120. The subsequent heating to liquefy and extract the fugitive ink may entail warming the printhead / nozzle 140,120 to room temperature or above, and / or to another suitable temperature, such as the curing temperature of the support matrix 104. After the fugitive or sacrificial ink is removed, the open vessels 102a of the interconnected three- dimensional network 112 may be perfused with blood, a blood substitute, a bodily fluid, or a non-biological fluid, such as a liquid metal. In some examples, the shell ink 118 and / or the support matrix 104 may comprise fugitive or sacrificial microparticles, that is, microparticles that are ultimately removed. Accordingly, the method may further comprise, after forming the interconnected three-dimensional network 112 of the core- shell filaments 102, removing the fugitive or sacrificial microparticles, thereby incorporating pores into the shell 108 and / or thesupport matrix 104. Removal of the sacrificial microparticles may occur simultaneously with or subsequently to removal of the fugitive or sacrificial ink, as described above.

[0038] The support matrix 104, the core ink 116 (or the elongated core 106), and / or the shell ink 118 (or the shell 108) may have a granular, continuous, and / or porous structure. Also or alternatively, the support matrix 104, the core ink 116 (or the elongated core 106), and / or the shell ink 118 (or the shell 108) may comprise a protein, a polymer, and / or one or more cells that may have the form of single cells, cell aggregates, and / or cell spheroids. The support matrix 104 in particular may be cellular, acellular, or synthetic. A cell concentration in the support matrix 104, in the core ink 116 (or the elongated core 106), and / or in the shell ink 118 (or the shell 108) may be at least about 100 cells / ml, at least about 1000 cells / ml, at least about 104cells / ml, at least about 105cells / ml, at least about 106cells / ml, at least about 107cells / ml, or at least about 108cells / ml, and / or no higher than about 109cells / ml, or no higher than about 108cells / ml.

[0039] The one or more cells may include fibroblasts, chondrocytes, osteoblasts, tendon cells, mast cells, wandering cells, immune cells, pericytes, inflammatory cells, endothelial cells, myocytes (cardiac, skeletal and smooth muscle cells), adipocytes (i.e., lipocytes or fat cells), parenchyma cells (neurons and glial cells, nephron cells, hepatocytes, pancreatic cells, lung parenchyma cells) and non-parenchymal cells (e.g., sinusoidal hepatic endothelial cells, Kupffer cells and hepatic stellate cells), hematopoietic stem cells (adult stem cells; i.e., hemocytoblasts) from the bone marrow that give rise to red blood cells, white blood cells, and platelets; mesenchymal stem cells (adult stem cells) from the bone marrow that give rise to stromal cells, fat cells, and types of bone cells; epithelial stem cells (progenitor cells) that give rise to the various types of skin cells; neural stem cells and neural progenitor cells that give rise to neuronal and glial cells; and / or muscle satellite cells (progenitor cells) that contribute to differentiated muscle tissue.

[0040] The protein and / or the polymer may include collagen type I, collagen type IV, laminin, fibronectin, fibrin, fibrinogen, gelatin, gelatin methacrylate, hyaluronic acid, hyaluronic acid methacrylate, chondroitin, aggrecan, fibroin, poly(ethylene glycol), hydrogel, alginate, alginate methacrylate, silk, poly(vinyl alcohol), poly-lysine, poly-omithine, agarose, agarose methacrylate, basement membrane extract (Matrigel), transglutaminase, siliconerubber, thermoplastic elastomer, polyolefin and polydiene elastomer, poly(vinyl chloride), natural rubber, heparinized polymer, polypeptide elastomer, and / or a liquid-crystal elastomer.

[0041] In some examples, the core ink 116 and / or the shell ink 118 may comprise an electrically conductive material (e.g., a liquid metal), an optically transparent dielectric material, or another material having a desired functionality and which can be flowed through the nozzle for printing. It is also contemplated that the core ink 116 and / or the shell ink 118 may include polymer fibers or mats, such that, upon printing of the core- shell filaments 102, the elongated cores 106 and / or the shells 108 are structurally reinforced with the polymer fibers or mats.

[0042] The core-shell nozzle 120 is not limited to just two channels (the core and shell channels 116,118). In some examples, the core-shell nozzle 120 may include one or more middle channels between the core channel 116 and the shell channel 118. In these examples, printing the core-shell filaments 102 may comprise flowing one or more additional inks through the one or more middle channels, and forming one or more middle layers between the elongated core 106 and the shell 108 of the core-shell filaments 102.

[0043] Depending on the intended application for the functional construct 100, which may in some examples be a tissue construct, the method may entail, during or after printing, modifying the support matrix 104 and / or the interconnected three-dimensional network 112 of the core-shell filaments 102 with light, heat, chemical(s), enzyme(s), and / or coating(s). In one example, the core-shell nozzle 120 itself may be configured to modify the core ink 116 and / or the shell ink 118 during flow therethrough by exposure to a predetermined temperature, a change in temperature, one or more chemicals, electrical energy, and / or a biological mechanism during printing. For example, curing of the shell 108 and / or liquefication and removal of the core 106 may be effected by exposure to a suitable elevated temperature. In another example, a polymeric layer may be applied during or after printing to encapsulate the core- shell filaments (e.g., to provide mechanical support or other protection). The polymeric layer may be applied as a conformal coating on individual core- shell filaments 102 or as an encapsulant over part or all of the interconnected three-dimensional network 112 of the filaments 102. As indicated above, the functional construct 100 may in some examples be a tissue construct that can be used as a tissue model for therapeutic applications, drug toxicity studies, drug screening, or disease modeling. In other examples the tissue construct may beused for suturing into a body or maturation in vitro. Alternatively, the functional construct may be used for robotics or other applications.

[0044] EXAMPLES

[0045] Here, examples of the above-described method for printing hierarchical branching vascular networks within soft and living matrices are described. Biomimetic vessels are embedded into granular hydrogel matrices or bulk cardiac tissues via particular implementations of the printing method described above, which may be referred to as coaxial embedded printing (co-EMB3DP) or coaxial sacrificial writing into functional tissues (coSWIFT), respectively. Each implementation relies on an extended core-shell printhead that promotes facile interconnections between printed branching vessels. Through careful optimization of multiple core-shell inks and matrices, it is demonstrated that embedded biomimetic vessels possessing a smooth muscle cell-laden shell surrounding perfusable lumens can be printed. Upon seeding these vessels with a confluent layer of endothelial cells, they exhibit good barrier function. As a final demonstration, biomimetic vascularized cardiac tissues composed of a densely cellular matrix of cardiac spheroids derived from human induced pluripotent stem cells are constructed. Importantly, these co-SWIFT cardiac tissues mature under perfusion, beat synchronously, and exhibit a cardio-effective drug response in vitro. This advance opens new avenues for the scalable biomanufacturing of organ- specific tissues for drug testing, disease modeling, and therapeutic use.

[0046] A key aspect of the technology is development of a printhead comprising a coreshell nozzle with an extended core tip. The extended core tip facilitates puncturing through the shell to enable connections to the elongated cores of other printed filaments, an advance which facilitates the printing of hierarchically branching, vascular networks. Also, a series of core and shell inks have been developed to identify preferred rheological properties for co-EMB3DP and co-SWIFT printing.

[0047] Coaxial printhead with extended core-shell nozzle

[0048] A coaxial printhead comprising an extended core-shell nozzle with two independently controllable fluidic channels for core and shell inks has been developed, as shown in FIGS. 3 A and 3B. This customized printhead is designed in Solidworks and built using a digital light projection lithography printing. The use of long needles to form the channels ensures minimal disruption when printing biomimetic vascular networks deep within acellular and densely cellular matrices. The shell ink first travels into an equilibration chamber,which provides a uniform pressure to facilitate ink flow through the shell channel. To ensure the shell ink is extruded uniformly, the height and diameter of the equilibration chamber are designed to be roughly one order of magnitude larger (2 mm) than the thickness of the shell channel (0.16 mm). If this criterion is not met, the ink may preferentially flow on one side of the shell channel.

[0049] Creating hierarchically branching core- shell filament networks may entail both branching from and reconnecting to an existing printed core-shell filament. The tubular inner wall of the core-shell nozzle may be extended up to or beyond 250 pm from (that is, downstream from) the tubular outer wall of the nozzle. This distance may correspond roughly to the thickness of the shell, which may be important for ensuring that the core-shell nozzle is able to puncture the shell to connect the cores of parent and daughter vessels (filaments) when performing branching and reconnection maneuvers, as shown in FIGS. 4A-4D.

[0050] Optimizing core ink, shell ink, and support matrix rheology

[0051] Multiple core and shell inks and support matrix materials have been developed to determine preferred material properties for co-EMB3D printing. First, granular alginate particles were produced via an in-air fluidic assembly method. Next, three transparent support matrices were produced to enable direct visualization of the co-EMB3DP process and their rheological properties and 3D structure were assessed by confocal imaging. Each support matrix is composed of granular particles of varying alginate concentration (0.5-2% alginate) and total particle volume fraction (<]> = 0.80-0.86). The granular alginate matrix (0.5% alginate and ([) = 0.86) with an intermediate shear yield stress (ry) of ~70 Pa and the desired viscoplastic and self-healing behavior is believed to be optimal for co-EMB3DP. A sacrificial gelatin core ink was then formulated such that its shear thinning behavior and ry~ 50 Pa nearly matched that of the granular alginate matrix. Inspired by the native vessel wall, which is predominantly composed of collagen, three shell inks were created using high-density collagen blended with either gelatin or PBS with ryvalues roughly an order of magnitude greater (ry~ 750 Pa), matched (ry~ 40 Pa), or an order of magnitude lower (ry~ 5 Pa) than the alginate support matrix. COMSOL simulations of each shell ink were carried out for flow through a coaxial printhead. Each core-shell ink combination could be successfully printed in the vertical direction within this support matrix, as can be seen from FIGS. 5A-5D. However, only the core-shell ink combination with the highest shell ry~ 750 Pa exhibited a uniform core-shell architecture when coaxially printed in the horizontal direction, as can be observed from FIGS.6A-6D. When the ryof the shell ink matches that of the matrix, the shell layer thins around the bottom of the filament. When its ryis less than the matrix, the shell does not entirely wrap around the core ink as needed to form the desired core-shell architecture. Hence, it is concluded that an optimal core-matrix ryratio of essentially unity and optimal shell-to-matrix ryratio of roughly 10 is beneficial if not required for coaxial embedded printing. Rheological properties of the core ink, shell inks (stiff, soft, and matched), and the support matrix are summarized in FIGS. 7 A and 7B.

[0052] Next, the effects of key printing parameters were explored by creating a symmetrical 2D vascular network via co-EMB3DP within the transparent alginate support matrix. To emulate native vasculature, coaxial vessels of varying diameter were printed with three generations of branching features (see FIG. 8) that obey Murray’s law:

[0054] where rP= radius of the parent vessel (filament) and rci= radius of the daughter vessels (filaments) branching from the parent vessel. Cross-sectional images of the printed vessels (see FIGS. 9A-9D) reveal that they retain their concentric core-shell architecture across each generation. To produce vessels with total diameters ranging from larger than 3 mm to smaller than 1 mm, the printing speed may varied, e.g., in this example, from 0.25 mm sec'1to 4 mm sec'1, while extruding the core and shell inks at a constant volumetric flow rate. Concomitantly, the core (luminal) diameter decreased from 1.57 mm to 0.29 mm, respectively, over these printing conditions, as can be seen in FIG. 10. Alternatively, at a constant printing speed, one can vary the core-to-shell ratio by changing the relative volumetric flow rates of each ink to produce core-to-shell ink ratios ranging from 0 to 1 in the printed filaments.

[0055] Embedding biomimetic vascular networks in collagen matrices

[0056] To further demonstrate co-EMB3D printing, a 3D hierarchical, branching vascular network embedded within an extracellular matrix including sacrificial microparticles and collagen (“pPOROS” collagen) was designed, printed, and perfused. This support matrix can be produced by suspending sacrificial gelatin-chitosan microparticles in a pre-polymer collagen solution followed by jamming to induce the desired shear thinning response when locally yielded at an applied shear stress (r) that exceeds ry~ 10 Pa. The pPOROS support matrix and collagen shell ink may be held below their gelation temperature for the duration of printing by pumping ice-cold water through a cooling system in the print gasket. The embedded core- shell filaments are arranged in a hierarchically branching network that is patterned in threedimensions and conforms to Murray’s law, as shown in FIG. 2. After printing, the tissue construct is warmed to 37 °C to facilitate collagen gelation and crosslinking in both the shell ink and pPOROS matrix, while simultaneously melting the sacrificial gelatin core, as shown in FIG. 11 A. The vascularized tissue construct is then perfused with a dye (PBS) as illustrated in FIG. 1 IB to visualize the perfused, interconnected luminal network.

[0057] To further enhance the physiological relevance, a biomimetic vascular network composed of a SMC-laden shell ink surrounding a sacrificial gelatin core ink was printed within this pPOROS matrix as illustrated in FIG. 12. Upon heating to 37 °C, the sacrificial core ink liquefies and the SMC-laden shell ink gels to create the blood vessel walls, which surround the interconnected luminal network that forms upon removal of the liquefied core ink. The luminal surfaces are coated with 1% v / v Matrigel on day 2 of perfusion prior to seeding the vessels with endothelial cells. After day 7 of perfusion, the smooth muscle cells remain viable, spread, and wrap around the vessel walls circumferentially akin to the morphology found in the native medial layer (see FIG. 13). The endothelial cells are arranged in a confluent monolayer with adherent junctions, as can be seen from the confocal images of FIGS. 14A and 14B, which show the branched, endothelialized vessel network produced by co-EMB3DP. A Miles assay was carried out to assess their barrier function. A three-fold decrease in dye diffusion from blood vessels that possess a confluent endothelium was observed compared to the bare (control) vessels, as indicated in FIG. 15.

[0058] Embedding biomimetic vascular networks in functional cardiac tissues

[0059] As a final demonstration, bulk cardiac tissues with biomimetic vasculature were generated via co-SWIFT. Hundreds of thousands of cardiac organ building blocks (OBBs) composed primarily of hiPSC-derived cardiomyocytes were created following previously reported protocols. Next, these cardiac OBBs were suspended in a fibrin solution that exhibits a fluid-like response under ambient conditions. Next, centrifugation was used to jam the cardiac OBB solution into a densely cellular matrix (ry~ 10 Pa, cell density of ~200xl06cells mL'1) Biomimetic vessels within this cardiac tissue matrix were patterned via co-SWIFT printing of the SMC-shell / gelatin sacrificial core inks. Thrombin, which is added into the shell ink, rapidly gels the fibrin solution surrounding the cardiac OBBs after printing. Upon warming the bulk cardiac tissue to 37°C, the sacrificial gelatin ink which forms the core melts, allowing the seamless removal of the core ink to form an interconnected luminal network. After one day of perfusion, a live-dead assay was carried out on these co-SWIFT cardiac tissues (overalldiameter = 2.8 mm and height = 1 cm), which revealed their high cell viability throughout their cross-section (see FIG. 16A). On day 2 of perfusion, endothelial cells were seeded onto the luminal surface of the embedded vessels. After day 7 of perfusion, the embedded vessels include a confluent layer of endothelial cells surrounded by smooth muscle cells, as can be seen in FIG. 16B. These densely cellular, vascularized cardiac tissues begin to contract on the first day of perfusion, while their contractile response increases by roughly three-fold from day 1 to day 5 of perfusion, as indicated in FIG. 17A. Importantly, these co-SWIFT cardiac tissues also exhibit a cardio-effective drug response. Upon perfusion of oxygenated media supplemented with isoproterenol at a concentration of 10 pM, their spontaneous beat frequency doubles. By contrast, the perfusion of media that contains 10 pM blebbistatin arrests beating of these cardiac tissues (see FIGS. 17B and 17C).

[0060] To highlight co-SWIFT’s promise for personalized biomanufacturing, a scaled model of the main branches for a patient-specific, left coronary artery (LCA) model was printed. To aid visualization, the initial branch and full arterial structure were simultaneously printed into both the transparent alginate matrix and densely cellular, cardiac OBB matrix Future plans include generating self-assembled microvascular networks (capillaries) within these co-SWIFT cardiac tissues and promoting their anastomosis to printed vessels in vitro to better recapitulate the native myocardium and enhance function.

[0061] Conclusions

[0062] In summary, printing methods for embedding biomimetic vascular networks into both acellular and densely cellular tissue matrices have been demonstrated. To demonstrate broad applicability, the rheological properties of core inks, shell inks, and support matrix have been engineered for co-EMB3DP in a granular alginate matrix and microporogen- structured collagen as well as co-SWIFT printing in functional cardiac tissues. Through the design, fabrication, and implementation of customized extended core-shell nozzles, hierarchical branching vessels composed of smooth muscle cell-laden shell ink that surround a sacrificial core ink can be produced. Such networks possess interconnected lumens (upon removing their sacrificial core), which can be wrapped by smooth muscle cells and seeded with endothelial cells to form a confluent endothelium that provides good barrier function. Finally, thick cardiac tissues with embedded biomimetic vessels have been created, whose design is guided by patient-specific data. This work provides an enabling advance for embedding biomimetic vascular networks within soft and living tissue constructs.

[0063] Experimental Section

[0064] Core-shell nozzle design and fabrication

[0065] The extended core-shell bioprinting nozzle was designed in Solidworks (Dassault Systemes) and printed on the EnvisionTec D4K printer using HTM140 resin (Desktop Metal). The nozzles were cleaned by connecting a syringe to the luer-lock and purging the fluid paths with 2-propanol (Sigma- Aldrich). A core nozzle (inner diameter = 0.25 mm, outer diameter = 0.52 mm, and length = 3.15 cm) was mated with a shell nozzle (inner diameter = 0.84 mm, outer diameter = 1.27 mm, and length = 1.9 cm) (Nordson EFD). The core nozzle was secured by injecting superglue (Loctite), while the shell nozzle was affixed using epoxy (Loctite).

[0066] Core-shell inks

[0067] Sacrificial gelatin used for the core and shell inks was prepared by dissolving 300 g Bloom type A gelatin (Sigma-Aldrich) at 15% w / v and stirred at 85 °C for 12 h. This gelatin stock was then adjusted to pH 7.4 using 1 N sodium hydroxide (Sigma- Aldrich). The gelatin was then sterile filtered and stored at 4 °C for up to 6 months. To prepare the core and shell inks, stock 15% w / v gelatin and 70 mg mL'1neutral collagen (LifeInk220, Advanced Biomatrix) respectively were diluted with different amounts of phosphate buffered saline with calcium and magnesium (PBS) (Corning) to the final concentrations in Table 1. To provide visual contrast between the core and the shell inks, either red pigment (Gamblin) or red food coloring (Ward’s Science) was added to the core inks.Table 1. Composition of co-SWIFT Materials

[0068] Granular alginate matrices

[0069] Alginate solutions were generated by dissolving medium viscosity alginic acid sodium salt (Sigma- Aldrich) in deionized water. Granular alginate particles (diameter = 190 pm ± 19.2 pm) were fabricated by injecting 0.5% alginate or 2% alginate solution from a lavender 45° nozzle (Nordson EFD) at a flow rate of 300 pL min'1into a 2 psi air stream controlled by a pressure box (Nordson EFD) through a purple 0.5 in nozzle (Nordson EFD). The alginate droplets were deposited into a gelation bath containing 100 mM CaCh (Sigma-16SUBSTITUTE SHEET (RULE 26)Aldrich) and 5% ethanol (KOPTEC), where they were then crosslinked for 1-3 h prior to being washed and stored in an aqueous salt solution containing 2 mM CaCh. These granular alginate particles were stored at 4 °C for up to 3 months before use.

[0070] To create printable matrices, the granular alginate particles were first swelled in PBS for 90 min and then centrifuged at 30g for 3 min. The supernatant was removed and the jammed particles were mixed with a serological pipette prior to loading them into the printing chamber or onto a controlled- shear rheometer. To quantify the volume fraction of alginate particles within these printable (jammed) matrices, 0.05% w / v 2 MDa TRITC-dextran (Thermo-Fisher) was added to the alginate solution prior to its consolidation. Confocal microscopy (Zeiss) coupled with image analysis was used to determine the volume fraction of granular alginate particles within the printable matrices. In addition, videos were generated from z-stack confocal images using a custom MATLAB script (MathWorks).

[0071] iPOROS matrices

[0072] The pPOROS matrix includes sacrificial gelatin microparticles and prepolymer collagen I. Following a previously published protocol, sacrificial microparticles were generated by dissolving 2% w / v gelatin type A (Sigma- Aldrich), 0.25% w / v Pluronic F-127 (Sigma- Aldrich), and 0.1% w / v chitosan (Sigma- Aldrich) in 51% v / v ethanol (Sigma- Aldrich) while stirring at 45 °C. The pH was adjusted to 6.32 using 1 N NaOH. The sacrificial gelatin microparticles were removed from heat and stirred overnight. The next day, the microparticles were homogenized mechanically and washed 3x in PBS. The microparticles (~30-50 pm in diameter) were stored at 4 °C for up to 4 months before use. Immediately prior to co-SWIFT printing, the sacrificial gelatin microparticles were centrifuged at 2000g for 3 min and the supernatant was removed. The particles were resuspended in 5 mg mL'1type I collagen (Advanced Biomatrix), transferred to 10 mL syringes, and centrifuged at 3000g for 5 min at 4 °C. The supernatant was removed and the pPOROS matrix was passed between two syringes using a syringe coupler 20 times to homogenize the matrix. The pPOROS matrix was stored in ice-water until it was used for printing or rheological characterization.

[0073] Rheological characterization

[0074] All rheological measurements were carried out on a controlled stress-controlled rheometer (DHR-3, TA Instruments) with a 25 mm diameter aluminum parallel plate geometry with 60 grit sandpaper attached to the surface to prevent slipping. Gap heights of 250 pm, 1 mm, and 2 mm, and were used for the inks, pPOROS matrix, and granular alginate matrix,respectively. Shear and oscillatory measurements for both inks and the pPOROS matrix were carried out at 2 °C, while measurements on the granular alginate matrix were performed at 20 °C. Apparent viscosity curves were collected by performing flow sweeps at shear rates ranging from 10 s'1to 0.001 s’1, while oscillatory measurements were performed at 0.5 Hz from 0.005 Pa until yielding.

[0075] Primary cell culture

[0076] Primary human umbilical vein endothelial cells (Lonza) and aortic smooth muscle cells (Cell Systems) were cultured in endothelial growth medium (EGM-2, Lonza) and VascuLife smooth muscle cell medium (LifeLine Cell Technology) respectively. Medium was refreshed every other day until the cells were 80% confluent. The cells were passaged by first rinsing with PBS without calcium and magnesium (PBS- / -) (Coming), then adding one quarter culture volume of 0.05% trypsin / EDTA (Gibco) to the flask for 4 min at 37 °C. The 0.05% trypsin / EDTA was quenched using DMEM / F12 with 10% fetal bovine serum (FBS) (Gibco). The cells were then centrifuged at 220g for 3 min. The supernatant was removed and the cells were split into preprepared flasks at a ratio ranging from 1:3 to 1:5. All primary cells were used from passage 4 to passage 7.

[0077] Embryoid body formation

[0078] BJFF iPSCs (provided by S. Jain at Washington University) were cultured on stemcell qualified growth factor reduced Matrigel (Coming) in mTeSR Plus stem cell medium (STEMCELL Technologies) in a 37 °C / 5% CO2 incubator. Once colonies reached 70-80% confluency, they were rinsed once in PBS- / -. ReLeSR (STEMCELL Technologies) was added to the flask and immediately aspirated away. The cells were transferred to the incubator for 7 min, before they were gently rinsed with culture medium and added to a freshly prepared flask at a ratio of 1:8. The iPSCs were lifted from the flask to form embryoid bodies (EBs) using the same method as for passaging, but were seeded in mTeSR Plus medium supplemented with 10 pM Y27632 (BioGems) into a non-adherent T25 flask (Coming) at a ratio of 112.5 cm2adherent culture area per non-adherent T25 flask on day -5 of differentiation. The flasks were then placed on an orbital shaker at 55 RPM. Medium was changed the next day with fresh mTeSR Plus without Y27632, then every other day until day 0.

[0079] Cardiac building blocks

[0080] A modified previously published protocol was used to differentiate the EBs into cardiac spheroids. On day 0, differentiation was initiated by adding cardiac differentiationmedium (CDM) composed of RPMI 1640 (Gibco) and 2% B27 without insulin (Gibco) and supplemented with 5 pM CHIR99021 (BioGems). The same medium was refreshed on day 1. On day 2 of differentiation, CDM with CHIR99021 was removed and replaced with CDM. On days 3 and 4, CDM with 2 pM iWRl (BioGems) was added. On day 5, the cells were cultured in CDM until beating was observed (day 6 or 7), after which CDM was replaced with cardiac maturation medium (CMM) composed of RPMI 1640 and 2% B27 with insulin (Gibco) and refreshed daily until the cardiac spheroids were used for co-SWIFT experiments (day 10-12).

[0081] Print path generation

[0082] Complex print paths were first designed in Solidworks and then exported to MATLAB. To generate the print path for the patient derived left coronary artery geometry, the structure was first downloaded from the NIH 3D print exchange and imported into Solidworks. A custom MATLAB script was used to translate the point data into G-code with the desired flow rates and print parameters, and the geometry scaled as needed. All other print paths were generated directly in G-code. Each print path was imported to A3200 motion control software (Aerotech) used to control our customized, multi-material 3D bioprinter.

[0083] Printing and. perfusion chambers

[0084] To facilitate coaxial printing and perfusion of embedded vasculature, customized chambers were either machined from polycarbonate (McMaster-Carr) or printed via stereolithography using BioMed Clear resin (Formlabs). In both cases, a watertight seal was formed using O-rings (McMaster-Carr), which were compressed using laser-cut acrylic plates (McMaster-Carr). The metal inlet and outlet pins (Nordson EFD) were epoxied (Loctite) to the main body of the culture chamber. The compliant spring arms were printed using EnvisionTec D4K printer with a HTM140 resin. Before sterilization, the compliant support arms were inserted into the gasket. The culture chambers and compliant support arms were autoclaved, while the acrylic windows were sterilized in 70% ethanol for a minimum of 30 min before use.

[0085] co-SWIFT printing implementation

[0086] The night before printing, 300 g Bloom type A gelatin which was dissolved at 15% w / v 70 °C for 1 h was diluted to 5% w / v using DMEM / F12 with HEPES (Gibco) and supplemented with CaCh to a final concentration of 2.5 mM and 5 U mL'1thrombin. A 3D printed mold in the desired shape of the tissue was coated with 10% Pluronic F-127 (Sigma- Aldrich) and inserted into the gasket. The gelatin- thrombin solution was used to fill the gasket around the mold. The gaskets were then stored at 4 °C overnight. On the day of printing, themolds were removed from the gelatin and the negative cavity was rinsed 3x with PBS. The chambers were then held at 4 °C.

[0087] Immediately prior to printing, an anchor gel which was used to affix the co-SWIFT tissue to the perfusion pin was prepared from two precursor solutions to prevent premature polymerization. Part 1 of the precursor solution contained 20 mg mL'1fibrinogen (Merck) diluted in DMEM / F12 with HEPES. Part 2 of the precursor solution consisted of 2.5 mM CaCh, 0.5 U mL'1thrombin, and 20 mg mL'1transglutaminase (Moo Gloo TI). Part 1 and part 2 were mixed in equal volume and allowed to polymerize at the base of the inlet pin. The extracellular matrix (ECM) gel, which provided immediate structural support for the coSWIFT tissue upon crosslinking after printing, consisted of 10 mg mL'1fibrinogen and 2.5 mM CaCh (Sigma- Aldrich) diluted in DMEM / F12 with HEPES.

[0088] Once the anchor gel was added to the chamber, the cOBBs were rinsed with 3:1 v / v ECM gel, centrifuged at 30g, and the supernatant was aspirated. The cOBBs were resuspended in 1:1 v / v ECM gel to cOBBs and transferred to a 1 mL disposable syringe (BD Biosciences). The cOBBs were centrifuged at 100g for 3 min and the supernatant removed. The resulting jammed cOBBs were then dispensed into the mold using an olive nozzle (inner diameter = 1.54 mm, Nordson EFD). Embedded vascular networks were rapidly printed within cOBB matrices via co-SWIFT of the sacrificial core ink and matched shell ink filled with SMCs. The customized printing and perfusion chambers were then transferred to the incubator at 37 °C to promote rapid polymerization of the collagen and fibrin within the shell ink and ECM gel, respectively, while the sacrificial gelatin ink in the core and the surrounding chamber liquify. After 20 min, the co-SWIFT tissues were connected to a peristaltic pump (MasterFlex), and the co-SWIFT medium consisting of equal volumes of CMM and vessel co-culture medium with 1:250 aprotinin (EMD Millipore) and lx Antibiotic-Antimycotic (Gibco) was used to evacuate the sacrificial gelatin from the vessel and chamber at a flow rate of 100 pL min'1. Once the sacrificial gelatin was removed, the flow rate was slowly increased to 250 pL min'1until day 2 when the tissues were endothelialized (as described below), and the flow rate was increased to 500 pL min'1for the duration of culture. The co-SWIFT medium was refreshed every other day.

[0089] Endothelialization of co-SWIFT vessels'. Vessels were coated with a 1% v / v Matrigel solution in either vessel co-culture medium or co-SWIFT culture medium for 2 h before endothelialization. HUVECs were lifted from the flask as previously described, theninjected into the vessel at 20xl06cells mL'1. These endothelial cells were allowed to attach for 80 min without flow at 37 °C during which the culture chamber was rotated 90° every 10 min to ensure even coating of the luminal surface. Flow was resumed at 50 pL min'1for 10 min, then slowly ramped up to its steady-state value of 500 pL min'1over a 20 min period.

[0090] Immunofluorescent staining and. confocal imaging

[0091] co-SWIFT vessels and cardiac co-SWIFT tissues were fixed in 4% paraformaldehyde (PFA) (Electron Microscopy Sciences) for 30 min or 45 min, respectively. Tissues were washed 3x for a minimum of 15 min in PBS before immunofluorescent staining. Permeabilization and blocking were performed for two hours in PBS containing 0.125% Triton X (Sigma- Aldrich), 0.5% bovine serum albumin (BSA) (Miltenyi Biotech), and 2% donkey serum (Sigma- Aldrich). Primary antibodies [cTnT (ab45932), aSMA (ab7817), CD31(ab9498) (Abeam)] were added at 1:200 in PBS with 0.125% Triton X and 0.5% BSA at 4 °C for 12 to 24 h. The constructs were then washed 3x for a minimum of 15 min in PBS before secondary antibody incubation. Alexa Fluor Plus conjugated secondary antibodies (Invitrogen) and UEA- I conjugated with fluorescein (Vector Laboratories) were then added in PBS with 2% donkey serum either for 2 hours at room temperature or at 4 °C overnight. 4’,6-diamidino-2- phenylindole (DAPI) (Thermo Fisher) was added for 30 min at room temperature before the secondary antibodies were washed out in PBS 3x for 15 min. Constructs were imaged on an upright confocal microscope (Zeiss).

[0092] Cell viability assays

[0093] The viability of smooth muscle cells encapsulated in the shell ink was assessed by first removing the cell culture medium from the customized printing and perfusion chamber and then adding PBS with ethidium homodimer and calcein AM at lx working concentrations of 0.5 pL mL'1and 2 pL mL'1, respectively, based on manufacturer recommendations (Invitrogen). The tissues were incubated at 37 °C for 30 min before imaging on an upright confocal microscope using a lOx water immersion objective. Quantitative image analysis was performed using Imaris (Oxford Instruments). To quantify cell viability, the cardiac co-SWIFT tissues were removed from flow after 24 h and sectioned into cylinders (roughly 1 mm in height and 2.5 mm in diameter) in a chamber containing ice-cold, co-SWIFT culture medium. Next, 50% of this medium replaced with an equal volume of ethidium homodimer and calcein AM at a 2x working concentration. Hoechst solution (Invitrogen) was added at a final concentrationof 0.25 pF mL'1. The tissues were incubated at 37 °C for 30 min before imaging on a confocal microscope with a 5x non-immersion objective.

[0094] Barrier function assay

[0095] A Miles permeability assay was performed to assess barrier integrity of the endothelial monolayer on the surface of the co-SWIFT vessels. After 1 week of culture, a 1% w / v solution of Evan’s blue dye (Chem-hnpex International) was dissolved in PBS. It was diluted 1:9 in vessel co-culture medium (final dye solution). The final dye solution was perfused through the vessel for 20 min at a flow rate of 500 pL min'1before the vessel was flushed with PBS for 5 min at the same flow rate to remove excess dye from the lumen of the vessel. The vessel construct was removed from the culture chamber and weighed on an analytical balance. The construct was then dissolved in 200 pF formamide (G-Biosciences) for 48 h at room temperature to recover the dye. The absorbance at 630 nm was recorded on the SynergyHT plate reader (BioTek) and the values were normalized to the weight of the construct.

[0096] Cardio-ejfective drug response

[0097] On day 10 of culture, either isoproterenol (Sigma- Aldrich) or blebbistatin (Sigma- Aldrich) was delivered intraluminally to the co-SWIFT cardiac tissues at a concentration of 10 pM for 30 min. After 30 min, videos of the cardiac co-SWIFT tissues were collected on a VHX-2000 digital microscope (Keyence). The resultant videos were analyzed using open- source software (Tracker; https: / / physlets.org / tracker / ).

[0098] This disclosure also includes the following aspects:

[0099] A first aspect is directed to a method of fabricating a functional construct for biological, robotic or other applications, the method comprising: printing core-shell filaments into a support matrix, each core-shell filament comprising an elongated core surrounded by a shell; and interconnecting the core-shell filaments to form branching and merging structures, thereby forming an interconnected three-dimensional network of the core- shell filaments within the support matrix.

[0100] A second aspect is related to the method of the preceding aspect, wherein each branching and merging structure comprises a junction of two or more core-shell filaments, wherein, at each junction, the elongated cores of the two or more core-shell filaments are connected, and the shells of the two or more core- shell filaments are connected.

[0101] A third aspect relates to the method of any preceding aspect, wherein the branchingand merging structures have a geometry that is biomimetic, derived from a patient scan, or otherwise conceived.

[0102] A fourth aspect relates to the method of any preceding aspect, wherein printing the core- shell filaments comprises, for each core- shell filament: flowing a core ink and shell ink through a core-shell nozzle moving relative to the support matrix, the core-shell nozzle comprising a core channel surrounded by a shell channel, wherein the core ink is flowed through the core channel and the shell ink is flowed through the shell channel.

[0103] A fifth aspect relates to the method of the preceding aspect, wherein the core- shell nozzle further comprises: an inner wall surrounded by an outer wall, the inner wall being spaced apart from the outer wall by the shell channel and surrounding the core channel, wherein the inner wall extends longitudinally beyond the outer wall at an outlet end of the core-shell nozzle, thereby defining an extended core tip.

[0104] A sixth aspect relates to the method of the preceding aspect, wherein interconnecting the core-shell filaments comprises puncturing shells of the core-shell filaments with the extended core tip.

[0105] A seventh aspect relates to the method of the preceding aspect, wherein printing is paused prior to puncturing and is resumed after puncturing.

[0106] An eighth aspect relates to the method of any preceding aspect, wherein the extended core tip has a length comparable to or greater than a shell thickness.

[0107] A ninth aspect relates to the method of any preceding aspect, wherein the extended core tip has a length of least about 10 microns, at least about 50 microns, at least about 100 microns, at least about 200 microns, or at least about 500 microns, and / or as large as about 1 cm, or as large as about 1 mm.

[0108] A tenth aspect relates to the method of any preceding aspect, wherein each of the core- shell filaments has a shell thickness in a range from about 10 microns to 1 cm, and / or wherein each of the core- shell filaments has a core diameter in a range from about 10 microns to about 1 cm.

[0109] An eleventh aspect relates to the method of any preceding aspect, wherein the movement of the core-shell nozzle relative to the support matrix occurs in x-, y-, and / or z- directions, the printing being omnidirectional printing.

[0110] A twelfth aspect relates to the method of any preceding aspect, wherein each of the core channel and the shell channel is configured with a switching mechanism to control flow.

[0111] A thirteenth aspect relates to the method of the preceding aspect, wherein the switching mechanism comprises a solenoid valve or a rotary valve.

[0112] A fourteenth aspect relates to the method of any preceding aspect, wherein the coreshell nozzle is configured to modify the core ink and / or the shell ink during flow therethrough by exposure to a predetermined temperature, a change in temperature, one or more chemicals, electrical energy, and / or a biological mechanism.

[0113] A fifteenth aspect relates to the method of any preceding aspect, wherein a ratio of core diameter to shell thickness is varied during printing by altering a flow rate of the core ink into the core channel relative to a flow rate of the shell ink into the shell channel.

[0114] A sixteenth aspect relates to the method of the preceding aspect, wherein increasing the flow rate of the core ink into the core channel relative to the flow rate of the shell ink into the shell channel increases the ratio of core diameter to shell thickness, and wherein decreasing the flow rate of the core ink into the core channel relative to the flow of the shell ink into the shell channel decreases the ratio of core diameter to shell thickness.

[0115] A seventeenth aspect relates to the method of any preceding aspect, wherein a size of the core- shell filament is varied during printing by altering flow rates of the core and shell inks into the core and shell channels and / or by altering a print speed of the core- shell nozzle relative to the support matrix.

[0116] An eighteenth aspect relates to the method of any preceding aspect, wherein a flow rate of the core ink through the nozzle ranges from greater than 0 to 10 cl / s, and wherein a flow rate of the shell ink through the nozzle ranges from greater than 0 to 10 cl / s.

[0117] A nineteenth aspect relates to the method of any preceding aspect, wherein the core channel and the shell channel are coaxial, and / or wherein the elongated core and the shell are coaxial.

[0118] A twentieth aspect relates to the method of any preceding aspect, wherein the core channel and the shell channel are not coaxial, and / or wherein the elongated core and the shell are not coaxial.

[0119] A twenty-first aspect relates to the method of any preceding aspect, wherein the core- shell nozzle includes two or more of the core channels surrounded by the shell channel, and / or wherein the core-shell filaments include two or more of the elongated cores surrounded by the shell.

[0120] A twenty- second aspect relates to the method of any preceding aspect, wherein thecore channel and the shell channel have a profile selected from the group consisting of: circular, oval, elliptical, triangular, diamond- shaped, square, pentagonal, hexagonal, octagonal, starshaped, or irregular.

[0121] A twenty-third aspect relates to the method of any preceding aspect, wherein the core channel and the shell channel have different profiles, or wherein the core channel and the shell channel have the same profile.

[0122] A twenty-fourth aspect relates to the method of any preceding aspect, wherein the shell ink has a stiffness higher than that of the support matrix.

[0123] A twenty-fifth aspect relates to the method of any preceding aspect, wherein the core ink comprises a fugitive or sacrificial ink, and further comprising: after forming the interconnected three-dimensional network of the core- shell filaments, removing the fugitive or sacrificial ink, thereby replacing the core-shell filaments with open vessels.

[0124] A twenty- sixth aspect relates to the method of the preceding aspect, further comprising, after removing the fugitive or sacrificial ink, perfusing the open vessels of the interconnected three-dimensional network with blood, a blood substitute, or a bodily fluid.

[0125] A twenty- seventh aspect relates to the method of any preceding aspect, wherein the shell ink and / or the support matrix comprises fugitive or sacrificial microparticles, and further comprising: after forming the interconnected three-dimensional network of the core-shell filaments, removing the fugitive or sacrificial microparticles, thereby incorporating pores into the shell and / or the support matrix.

[0126] A twenty-eighth aspect relates to the method of any preceding aspect, wherein the support matrix, the core ink, the elongated core, the shell ink, and / or the shell has a granular, continuous, and / or porous structure.

[0127] A twenty-ninth aspect relates to the method of any preceding aspect, wherein the support matrix, the core ink, the elongated core, the shell ink, and / or the shell comprise a protein, a polymer, and / or one or more cells.

[0128] A thirtieth aspect relates to the method of the preceding aspect, wherein the one or more cells have the form of single cells, cell aggregates, and / or cell spheroids.

[0129] A thirty-first aspect relates to the method of any preceding aspect, wherein a cell concentration in the support matrix, the core ink, the elongated core, the shell ink, and / or the shell is at least about 100 cells / ml, at least about 1000 cells / ml, at least about 104cells / ml, at least about 105cells / ml, at least about 106cells / ml, at least about 107cells / ml, or at least aboutIO8cells / ml, and / or no higher than about 109cells / ml, or no higher than about 108cells / ml.

[0130] A thirty-second aspect relates to the method of any preceding aspect, wherein the one or more cells comprise: fibroblasts, chondrocytes, osteoblasts, tendon cells, mast cells, wandering cells, immune cells, pericytes, inflammatory cells, endothelial cells, myocytes (cardiac, skeletal and smooth muscle cells), adipocytes (i.e., lipocytes or fat cells), parenchyma cells (neurons and glial cells, nephron cells, hepatocytes, pancreatic cells, lung parenchyma cells) and non-parenchymal cells (e.g., sinusoidal hepatic endothelial cells, Kupffer cells and hepatic stellate cells), hematopoietic stem cells (adult stem cells; i.e., hemocytoblasts) from the bone marrow that give rise to red blood cells, white blood cells, and platelets; mesenchymal stem cells (adult stem cells) from the bone marrow that give rise to stromal cells, fat cells, and types of bone cells; epithelial stem cells (progenitor cells) that give rise to the various types of skin cells; neural stem cells and neural progenitor cells that give rise to neuronal and glial cells; and / or muscle satellite cells (progenitor cells) that contribute to differentiated muscle tissue.

[0131] A thirty-third aspect relates to the method of any preceding aspect, wherein the protein and / or the polymer comprise collagen type I, collagen type IV, laminin, fibronectin, fibrin, fibrinogen, gelatin, gelatin methacrylate, hyaluronic acid, hyaluronic acid methacrylate, chondroitin, aggrecan, fibroin, poly(ethylene glycol), hydrogel, alginate, alginate methacrylate, silk, poly(vinyl alcohol), poly-lysine, poly-ornithine, agarose, agarose methacrylate, basement membrane extract (Matrigel), transglutaminase, silicone rubber, thermoplastic elastomer, polyolefin and polydiene elastomer, poly(vinyl chloride), natural rubber, heparinized polymer, polypeptide elastomer, and / or a liquid-crystal elastomer.

[0132] A thirty-fourth aspect relates to the method of any preceding aspect, wherein the core ink and / or the shell ink comprises an electrically conductive material (e.g., a liquid metal), an optically transparent dielectric material, or another material having a desired functionality.

[0133] A thirty-fifth aspect relates to the method of any preceding aspect, wherein the core ink and / or the shell ink includes polymer fibers or mats, and wherein, upon printing of the coreshell filaments, the elongated cores and / or the shells are structurally reinforced with the polymer fibers or mats.

[0134] A thirty- sixth aspect relates to the method of any preceding aspect, wherein a polymeric layer applied during or after printing encapsulates the core-shell filaments to provide mechanical support.

[0135] A thirty-seventh aspect relates to the method of any preceding aspect, wherein thecore- shell nozzle includes one or more middle channels between the core channel and the shell channel, and wherein printing the core-shell filaments comprises, for at least one of the coreshell filaments: flowing one or more additional inks through the one or more middle channels, and forming one or more middle layers between the elongated core and the shell of the respective core-shell filaments.

[0136] A thirty-eighth aspect relates to the method of any preceding aspect, wherein the support matrix is cellular, acellular, or synthetic.

[0137] A thirty-ninth aspect relates to the method of any preceding aspect, further comprising, after printing, modifying the support matrix and / or the interconnected three- dimensional network of the core-shell filaments with light, heat, chemical(s), enzyme(s), and / or coating(s).

[0138] A fortieth aspect relates to the method of any preceding aspect, further comprising, after forming the interconnected three-dimensional network of the core-shell filaments, removing the support matrix.

[0139] A forty-first aspect relates to the method of any preceding aspect, wherein the functional construct is a tissue construct.

[0140] A forty- second aspect relates to the method of the preceding aspect, wherein the tissue construct is used as a tissue model for therapeutic applications, drug toxicity studies, drug screening, or disease modeling.

[0141] A forty-third aspect relates to the method of any preceding aspect, wherein the tissue construct is used for suturing into a body or maturation in vitro.

[0142] A forty-fourth aspect relates to a tissue or other functional construct produced by the method of any preceding aspect and / or including any feature(s) recited by any preceding aspect.

[0143] To clarify the use of and to hereby provide notice to the public, the phrases "at least one of , , ... and <N>" or "at least one of , , ... <N>, or combinations thereof" or ", , ... and / or <N>" are defined by the Applicant in the broadest sense, superseding any other implied definitions hereinbefore or hereinafter unless expressly asserted by the Applicant to the contrary, to mean one or more elements selected from the group comprising A, B, ... and N. In other words, the phrases mean any combination of one or more of the elements A, B, ... or N including any one element alone or the one element in combination with one or more of the other elements which may also include, in combination, additional 1elements not listed. Unless otherwise indicated or the context suggests otherwise, as used herein, "a" or "an" means "at least one" or "one or more."

[0144] While various embodiments have been described, it will be apparent to those of ordinary skill in the art that many more embodiments and implementations are possible. Accordingly, the embodiments described herein are examples, not the only possible embodiments and implementations.

Claims

CLAIMSWhat is claimed is:

1. A method of fabricating a functional construct for biological, robotic or other applications, the method comprising: printing core-shell filaments into a support matrix, each core-shell filament comprising an elongated core surrounded by a shell; and interconnecting the core- shell filaments to form branching and merging structures, thereby forming an interconnected three-dimensional network of the core-shell filaments within the support matrix.

2. The method of claim 1, wherein each branching and merging structure comprises a junction of two or more core-shell filaments, wherein, at each junction, the elongated cores of the two or more core-shell filaments are connected, and the shells of the two or more core- shell filaments are connected.

3. The method of claim 1, wherein the branching and merging structures have a geometry that is biomimetic, derived from a patient scan, or otherwise conceived.

4. The method of claim 1, wherein printing the core-shell filaments comprises, for each core- shell filament: flowing a core ink and shell ink through a core- shell nozzle moving relative to the support matrix, the core- shell nozzle comprising a core channel surrounded by a shell channel, wherein the core ink is flowed through the core channel and the shell ink is flowed through the shell channel.

5. The method of claim 4, wherein the core- shell nozzle further comprises: an inner wall surrounded by an outer wall, the inner wall being spaced apart from the outer wall by the shell channel and surrounding the core channel, wherein the inner wall extends longitudinally beyond the outer wall at an outlet end of the core- shell nozzle, thereby defining an extended core tip.

6. The method of claim 5, wherein interconnecting the core-shell filaments comprises puncturing shells of the core-shell filaments with the extended core tip.

7. The method of claim 6, wherein printing is paused prior to puncturing and is resumed after puncturing.

8. The method of claim 5, wherein the extended core tip has a length comparable to or greater than a shell thickness.

9. The method of claim 5, wherein the extended core tip has a length of least about 10 microns, at least about 50 microns, at least about 100 microns, at least about 200 microns, or at least about 500 microns, and / or as large as about 1 cm, or as large as about 1 mm.

10. The method of claim 1, wherein each of the core-shell filaments has a shell thickness in a range from about 10 microns to 1 cm, and / or wherein each of the core- shell filaments has a core diameter in a range from about 10 microns to about 1 cm.

11. The method of claim 1, wherein the movement of the core- shell nozzle relative to the support matrix occurs in x-, y-, and / or z-directions, the printing being omnidirectional printing.

12. The method of claim 4, wherein each of the core channel and the shell channel is configured with a switching mechanism to control flow through the respective channel.

13. The method of claim 12, wherein the switching mechanism comprises a solenoid valve or a rotary valve.

14. The method of claim 4, wherein the core-shell nozzle is configured to modify the core ink and / or the shell ink during flow therethrough by exposure to a predetermined temperature, a change in temperature, one or more chemicals, electrical energy, and / or a biological mechanism.

15. The method of claim 4, wherein a ratio of core diameter to shell thickness is varied during printing by altering a flow rate of the core ink into the core channel relative to a flow rate of the shell ink into the shell channel.

16. The method of claim 15, wherein increasing the flow rate of the core ink into the core channel relative to the flow rate of the shell ink into the shell channel increases the ratio of core diameter to shell thickness, and wherein decreasing the flow rate of the core ink into the core channel relative to the flow of the shell ink into the shell channel decreases the ratio of core diameter to shell thickness.

17. The method of claim 4, wherein a size of the core- shell filament is varied during printing by altering flow rates of the core and shell inks into the core and shell channels and / or by altering a print speed of the core-shell nozzle relative to the support matrix.

18. The method of claim 4, wherein a flow rate of the core ink through the nozzle ranges from greater than 0 to 10 cl / s, and wherein a flow rate of the shell ink through the nozzle ranges from greater than 0 to 10 cl / s.

19. The method of claim 4, wherein the core channel and the shell channel are coaxial, and / or wherein the elongated core and the shell are coaxial.

20. The method of claim 4, wherein the core channel and the shell channel are not coaxial, and / or wherein the elongated core and the shell are not coaxial.

21. The method of claim 4, wherein the core- shell nozzle includes two or more of the core channels surrounded by the shell channel, and / or wherein the core-shell filaments include two or more of the elongated cores surrounded by the shell.

22. The method of claim 4, wherein the core channel and the shell channel have a profile selected from the group consisting of: circular, oval, elliptical, triangular, diamondshaped, square, pentagonal, hexagonal, octagonal, star- shaped, or irregular.

23. The method of claim 4, wherein the core channel and the shell channel have different profiles, or wherein the core channel and the shell channel have the same profile.

24. The method of claim 4, wherein the shell ink has a stiffness higher than that of the support matrix.

25. The method of claim 4, wherein the core ink comprises a fugitive or sacrificial ink, and further comprising: after forming the interconnected three-dimensional network of the core- shell filaments, removing the fugitive or sacrificial ink, thereby replacing the core-shell filaments with open vessels.

26. The method of claim 25, further comprising, after removing the fugitive or sacrificial ink, perfusing the open vessels of the interconnected three-dimensional network with blood, a blood substitute, or a bodily fluid.

27. The method of claim 4, wherein the shell ink and / or the support matrix comprises fugitive or sacrificial microparticles, and further comprising: after forming the interconnected three-dimensional network of the core- shell filaments, removing the fugitive or sacrificial microparticles, thereby incorporating pores into the shell and / or the support matrix.

28. The method of claim 4, wherein the support matrix, the core ink, the elongated core, the shell ink, and / or the shell has a granular, continuous, and / or porous structure.

29. The method of claim 4, wherein the support matrix, the core ink, the elongated core, the shell ink, and / or the shell comprise a protein, a polymer, and / or one or more cells.

30. The method of claim 29, wherein the one or more cells have the form of single cells, cell aggregates, and / or cell spheroids.

31. The method of claim 29, wherein a cell concentration in the support matrix, the core ink, the elongated core, the shell ink, and / or the shell is at least about 100 cells / ml, at least about 1000 cells / ml, at least about 104cells / ml, at least about 105cells / ml, at least about106cells / ml, at least about 107cells / ml, or at least about 108cells / ml, and / or no higher than about 109cells / ml, or no higher than about 108cells / ml.

32. The method of claim 29, wherein the one or more cells comprise: fibroblasts, chondrocytes, osteoblasts, tendon cells, mast cells, wandering cells, immune cells, pericytes, inflammatory cells, endothelial cells, myocytes (cardiac, skeletal and smooth muscle cells), adipocytes (i.e., lipocytes or fat cells), parenchyma cells (neurons and glial cells, nephron cells, hepatocytes, pancreatic cells, lung parenchyma cells) and non-parenchymal cells (e.g., sinusoidal hepatic endothelial cells, Kupffer cells and hepatic stellate cells), hematopoietic stem cells (adult stem cells; i.e., hemocytoblasts) from the bone marrow that give rise to red blood cells, white blood cells, and platelets; mesenchymal stem cells (adult stem cells) from the bone marrow that give rise to stromal cells, fat cells, and types of bone cells; epithelial stem cells (progenitor cells) that give rise to the various types of skin cells; neural stem cells and neural progenitor cells that give rise to neuronal and glial cells; and / or muscle satellite cells (progenitor cells) that contribute to differentiated muscle tissue.

33. The method of claim 29, wherein the protein and / or the polymer comprise collagen type I, collagen type IV, laminin, fibronectin, fibrin, fibrinogen, gelatin, gelatin methacrylate, hyaluronic acid, hyaluronic acid methacrylate, chondroitin, aggrecan, fibroin, poly(ethylene glycol), hydrogel, alginate, alginate methacrylate, silk, poly(vinyl alcohol), poly-lysine, poly-omithine, agarose, agarose methacrylate, basement membrane extract (Matrigel), transglutaminase, silicone rubber, thermoplastic elastomer, polyolefin and polydiene elastomer, poly(vinyl chloride), natural rubber, heparinized polymer, polypeptide elastomer, and / or a liquid-crystal elastomer.

34. The method of claim 4, wherein the core ink and / or the shell ink comprises an electrically conductive material, an optically transparent dielectric material, or another material having a desired functionality.

35. The method of claim 4, wherein the core ink and / or the shell ink includes polymer fibers or mats, and wherein, upon printing of the core-shell filaments, the elongated cores and / or the shells are structurally reinforced with the polymer fibers or mats.

36. The method of claim 1, wherein a polymeric layer applied during or after printing encapsulates the core-shell filaments to provide mechanical support.

37. The method of claim 4, wherein the core- shell nozzle includes one or more middle channels between the core channel and the shell channel, and wherein printing the core-shell filaments comprises, for at least one of the core-shell filaments: flowing one or more additional inks through the one or more middle channels, and forming one or more middle layers between the elongated core and the shell of the respective core- shell filaments.

38. The method of claim 1, wherein the support matrix is cellular, acellular, or synthetic.

39. The method of claim 1, further comprising, after printing, modifying the support matrix and / or the interconnected three-dimensional network of the core- shell filaments with light, heat, chemical(s), enzyme(s), and / or coating(s).

40. The method of claim 1, further comprising, after forming the interconnected three-dimensional network of the core-shell filaments, removing the support matrix.

41. The method of claim 1, wherein the functional construct is a tissue construct.

42. The method of claim 41, wherein the tissue construct is used as a tissue model for therapeutic applications, drug toxicity studies, drug screening, or disease modeling.

43. The method of claim 41, wherein the tissue construct is used for suturing into a body or maturation in vitro.

44. A tissue or other functional construct produced by the method of claim 1.

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