System and method for printing core-shell fiber

Abstract

To provide a system and method for fabricating core-shell fiber structures, including hollow fiber and multi-shell structures, and for fabricating three-dimensional (3D) structures from digital files.SOLUTION: A printhead for a three-dimensional printer includes a multi-channel enclosure including a core channel outlet, a first shell channel outlet, and a first fluid focusing chamber that converges toward a dispense channel. The core channel outlet is at a central region of the enclosure, and the core and shell channels extend to respective depths within the enclosure. A plurality of shell channels also includes an inner shell channel that extends a greater distance into the focusing chamber than an outer shell channel, and the core channel extends a greater distance into the focusing chamber than either shell channel. The core and first shell channels each have a separate fluid reservoir, input orifice and control valve.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to the filing dates of U.S. Provisional Patent Application No. 62 / 929,720, filed November 1, 2019, and U.S. Provisional Patent Application No. 63 / 030,885, filed May 27, 2020, the disclosures of both applications being incorporated herein by reference in their entireties.

[0002] The present invention relates to systems and methods for fabricating core-shell fiber structures and three-dimensional (3D) printing of such structures from digital files. In some embodiments, the printed fibers comprise living cells. [Background technology]

[0003] Tissue engineering techniques have long sought to create viable synthetic structures that can mimic and / or replace biological organs and tissues using a myriad of materials and methods. The lack of a pre-patterned vasculature is one of the major factors limiting the success of current tissue engineering strategies, and the current inability to create thick tissue constructs containing endogenous artificial vasculature or nutrient conduits that can integrate with host tissue is a major technical obstacle preventing the generation and / or transplantation of larger, viable, and / or metabolically active tissues.

[0004] 3D printing, a form of additive manufacturing, has been applied to create three-dimensional objects directly from digital files, where the object is built layer by layer to achieve the desired three-dimensional structure. Early efforts to adapt these 3D printing techniques for hollow vascular patterning have primarily focused on printing and subsequent removal of the sacrificial material. For example, Bertassoni et al. used a physical method to remove templated agarose from a cast around a photocrosslinkable acrylated hydrogel such as gelMA (Lab Chip 14:2202 (2014)). While the printed agarose fibers showed minimal binding to GelMA, manual removal was unfortunately required, which is time-consuming and difficult, and requires the cast hydrogel to be stronger than the agarose fibers.

[0005] An alternative approach involves printing a network of sacrificial fibers from a material that can be subsequently removed by solubilization or liquefaction. For example, Wu et al. printed a 3D perfusable vascular tree by extruding a sacrificial Pluronic® F127 filament into a Pluronic® F127-diacrylate gel reservoir to provide support during printing (Adv Mater. 2011;23:H178-183). After photocuring the surrounding acrylate-modified Pluronic® F127-diacrylate, the unmodified Pluronic® F127 conduit can be liquefied by lowering the temperature below the critical micelle temperature of Pluronic® F127, leaving a perfusable conduit. Using a similar approach, Lee et al. deposited a layer of collagen support matrix around gelatin containing human umbilical vein endothelial cells (HUVECs) (Biomaterials. 2014;35:8092-8102). After printing, the gelatin dissolved, helping to "activate" the cell seeding of HUVECs into the surrounding collagen. A variety of other sacrificial materials have also been printed, including the "carbohydrate glass" employed as a sacrificial material by Miller et al., demonstrating subsequent perfusion of hollow networks (Nat Mater. 2012;11:768-774).

[0006] However, to date, Pluronic® F127 has become the most commonly used sacrificial material due to its low-temperature liquefaction properties, and Kolesky et al. have successfully used it with various support materials to create thick, vascularized tissue constructs (Adv Mater. 2014;26:3124-3130) (co-printing channel structures of Pluronic F127 and cell-loaded gelatin-methacrylate (GelMA); Proc Natl Acad Sci USA. 2016;113:3179-3184) (Pluronic® F127 mixed with thrombin was designated a "vascular ink" for the indirect printing of sacrificial conduits within a cell-loaded gelatin-fibrinogen bioink). However, sacrificial materials, particularly Pluronic® F127, are cytotoxic at high concentrations, liquefied Pluronic® is unlikely to be completely removed from the hollow conduit, and the effect of liquefied Pluronic® on surrounding areas of tissue is unknown.

[0007] A more recent alternative to sacrificial hollow fiber patterning uses a focused beam of laser light to thermally ablate regions within a preformed (or printed) tissue construct. As the laser beam travels, it leaves behind hollow tunnels, making this technique relatively fast and capable of patterning branched hollow tubes in 3D with high resolution, potentially down to capillaries with diameters of 10-20 μm. The penetration depth of the beam can be increased by using two-photon laser light, which also serves to reduce the intensity of the light outside the focus, thereby reducing phototoxicity to the outer regions of the ablated conduit.

[0008] Direct bioprinting of hollow tubes within larger tissues has also been attempted. For example, Gao et al. demonstrated the use of a coaxial needle to generate and print hollow alginate fibers using a calcium chloride crosslinking solution at the alginate fiber core. The print nozzle was configured to create structures with internally perfusable microconduits through an internal flow of calcium solution and an external flow of alginate solution (i.e., bioink). In this technique, hollow microconduits were printed on a stage, which was then gradually lowered into a calcium bath for secondary crosslinking. (Biomaterials. 2015;61:203-215.) Hinton et al. developed an alternative to liquid immersion printing, employing an extrusion method to directly print structures using various hydrogels supported by a sacrificial gelatin microparticle bath to facilitate crosslinking. (Sci Adv. 2015;1:e1500758).

[0009] Unfortunately, the above-described systems, devices, and materials used in conventional 3D bioprinting of hollow fiber networks suffer from numerous drawbacks that prevent their more practical, effective, and widespread implementation. Manual (physical) removal of the above-described sacrificial materials is impractical, inconsistent, time-consuming, and perhaps impossible for small vessels. Furthermore, patterning blood vessels with sacrificial materials limits the ability to pattern cells and / or biomaterials axially surrounding hollow conduits. It is difficult to imagine using this technique to create, for example, patterned conduit networks that mimic the structure of real arterioles, with smooth muscle cells surrounding an inner layer of endothelial cells.

[0010] Laser ablation limits penetration depth to only 1-2 mm and requires an optically transparent material that does not scatter the beam, whereas most cellularized tissues are opaque and scatter light. Finally, in extrusion printing of sacrificial materials, the diameter of the sacrificial fiber (and subsequently the inner diameter of the conduit) is determined by the diameter of the extrusion needle. Because this diameter is fixed, there is no opportunity to dynamically change the lumen diameter of the conduit in different regions of the tissue.

[0011] Thus, there is a need for systems and devices that can dispense and pattern hollow conduits within 3D tissues by precisely positioning pro-angiogenic bioinks and various cell types axially and parallel to the hollow conduits. The required technology must be compatible with cell viability, and the inner diameter of the printed conduits must be dynamically variable, from capillaries to large vessels, within a single tissue construct. For example, it may be desirable to have large-diameter vessels at the opening of the tissue where the perfusion device is attached, and then reduce the lumen diameter to model smaller vessels inside the tissue. Varying vessel diameter can also be a useful tool for modulating flow alterations and restrictions in diseases such as atherosclerosis. The present invention addresses these and other unmet needs. Summary of the Invention

[0012] Aspects of the invention include systems and methods for fabricating core-shell fiber structures, including hollow fibers and multi-shell structures, and for fabricating three-dimensional (3D) structures from digital files. In some embodiments, the printed fibers contain living cells. As demonstrated herein, direct printing of core-shell fibers using the subject invention can produce fibers with various diameters and multiple shells, and can generate hollow vessels with multiple cell layers by loading different types of cells within the various shells with precise axial and parallel placement. Furthermore, the composition of the vessel wall (cell type and biomaterial composition) can be varied along the length of the channel during continuous printing.

[0013] An embodiment of the invention includes a microfluidic printhead for generating core-shell fiber structures, the printhead including a plurality of stacked, preferably bonded, layers forming a plurality of flow paths, including at least one core channel having at least one inlet 100 and outlet 102 and one or more fluidic switches, a first shell channel having at least one inlet and outlet, at least one multi-channel enclosure 108, and a dispensing channel 110, the multi-channel enclosure 108 including the core channel outlet 102, the first shell channel outlet 106, and a first fluid focusing chamber 112, the core channel outlet 102 being located in a central region of the multi-channel enclosure 108. Preferably, core channel outlet 102 extends a first vertical depth into first fluid focusing chamber 112 in alignment with dispense channel 110; first shell channel outlet 106 is concentrically disposed around the core channel and in fluid communication with the inlet of first fluid focusing chamber 112 and extends a second vertical depth into multichannel enclosure 108; first fluid focusing chamber 112 converges toward dispense channel 110; preferably, first fluid focusing chamber 112 comprises a frustoconical shape configured to focus fluid toward dispense channel 110. In some embodiments, first shell channel outlet 106 has a gradient width that increases with increasing depth into multichannel enclosure 108. In the exemplary embodiment, the first shell channel outlet 106 comprises a hollow cylinder having an axis of rotation that does not intersect with the core channel outlet 102 .

[0014] In some embodiments, the core channel outlet 102 extends through most of the length of the multi-channel enclosure 108. In preferred embodiments, the first vertical depth is greater than the second vertical depth, such that the core channel outlet 102 extends further into the multi-channel enclosure 108 and / or the first fluid focus chamber 112 than the first shell channel outlet 106. In alternative embodiments, the second vertical depth is greater than the first vertical depth, such that the first shell channel outlet 106 extends further into the multi-channel enclosure 108 than the core channel outlet 102. In some embodiments, the first fluid focus chamber 112 is disposed in a separate layer of the printhead, and the first shell channel outlet 106 extends from a previous layer of the printhead into the first fluid focus chamber 112 of an adjacent, downstream layer of the printhead.

[0015] In some embodiments, the print head comprises at least two core subchannels that converge to form a core channel outlet 102 in fluid communication with a first fluid focusing chamber 112. In preferred embodiments, the at least two core subchannels converge at or proximate to the core channel outlet 102, the multichannel enclosure 108, and / or a fluid distribution orifice, as described further herein. In particularly preferred embodiments, the at least two subchannels converge in an immediately preceding layer of the print head or in the same layer of the print head as the core channel outlet 102, the multichannel enclosure, and / or the fluid distribution orifice. In one embodiment, the core channels are configured to dispense a non-crosslinkable material. In an exemplary embodiment, the first core subchannel comprises a sheath fluid input orifice and a control valve, and the second core subchannel comprises a buffer input orifice and a control valve.

[0016] In some embodiments, the printhead further includes a second shell channel 128 having at least one inlet and outlet, the second shell channel outlet and / or inlet 132, 106 being concentrically disposed around the first shell channel outlet 106 of a multi-channel enclosure 108 in the same layer of the printhead and in fluid communication with the first fluid focus chamber 112. In preferred embodiments, the first shell channel outlet 106 extends further into the multi-channel enclosure 108 than the second shell channel outlet 132. In some embodiments, the first fluid focus chamber 112 is disposed in a separate layer of the printhead, and the first and / or second shell channel outlets 106, 132 extend from a previous layer of the printhead into the first fluid focus chamber 112 in an adjacent, downstream layer of the printhead.

[0017] In an alternative embodiment, the print head further includes a second shell channel 128 having at least one inlet and outlet, and a second multichannel enclosure 134 disposed between first fluid focusing chamber 112 and the distal end of dispense channel 110, said second multichannel enclosure 134 comprising said dispense channel 110, said second shell channel outlet 132, and a second fluid focusing chamber 136, said dispense channel 110 being disposed in a central region of second multichannel enclosure 134 and Preferably, second shell channel outlet 132 is concentrically disposed about dispense channel 110 and is in fluid communication with the inlet of second fluid focusing chamber 136 and extends a first vertical depth into the multichannel enclosure, second fluid focusing chamber 136 converging toward dispense channel 110, preferably second fluid focusing chamber 136 comprising a frustoconical shape configured to focus fluid toward dispense channel 110. In some embodiments, first and second multichannel enclosures 108, 344 are disposed in successive layers of the printhead. In further embodiments, the first multichannel enclosure and the second multichannel enclosure, or portions thereof, can be disposed in the same layer of the printhead, for example, the second multichannel enclosure can overlap the first fluid focusing chamber in the same layer of the printhead. In some embodiments, the second fluid focusing chamber 136 is disposed in a separate layer of the printhead, and the second shell channel outlet 132 extends from a previous layer of the printhead into the second fluid focusing chamber 136 in an adjacent downstream layer.

[0018] In a preferred embodiment, the first shell channel, the second shell channel 128, or both, further comprise at least one fluid distribution orifice configured to distribute fluid around the first shell channel outlet 106 and / or the second shell channel outlet 132. In one embodiment, the fluid distribution orifice connects the first and / or second shell channel inlet 104, 130 to the apex 116 of the upper curved surface 114 of the first and / or second shell channel outlet 106, 132, and preferably, the upper curved surface 114 of the first and / or second shell channel outlet 106, 132 has a parabolic or elliptical shape. In an exemplary embodiment, the first and / or second shell channel outlet 106, 132 comprises a truncated hollow cylinder with an elliptical upper surface, the apex 116 of which is located at the fluid distribution orifice.

[0019] In some embodiments, the first and / or second shell channel inlets 104, 130 are configured to dispense two different materials, including two different hydrogel materials and / or two different cell materials, such that the composition of the first and / or second shell layers can vary along the length of the printed fiber. In some embodiments, the first and / or second shell channel inlets 104, 130 include separate subchannels with separate fluid reservoirs, input orifices, and control valves. In some embodiments, the subchannels have the same fluid reservoirs, input orifices, and control valves. In other embodiments, the first and / or second shell channels can include two fluidic switches, and each subchannel can be fluidically connected to a separate fluidic switch. In further embodiments, the core channel can include two fluidic switches, and each core inlet subchannel can be fluidically connected to a separate fluidic switch.

[0020] In one embodiment, the first and / or second shell channel inlets 104, 130 include two or more shell inlet subchannels 126 with separate fluid reservoirs, input orifices, and control valves that converge to form a single shell channel outlet 106, 132 and / or fluid distribution orifice. In an exemplary embodiment, a softer hydrogel material flowing through one shell inlet subchannel 126 can be switched to a stiffer hydrogel material flowing through a second shell inlet subchannel to strengthen the fiber as needed. In another exemplary embodiment, a first cell-containing material in one shell inlet subchannel 126 can be switched with a second cell-containing material in a second shell inlet subchannel 126 to create an array of cell types along the length of the fiber.

[0021] In one embodiment, first and / or second shell channel inlets 104, 130 include three or more shell inlet subchannels 126 with separate fluid reservoirs, input orifices, and control valves that converge to form a single shell channel outlet 106, 132 and / or fluid distribution orifice. In a preferred embodiment, one of the three shell inlet subchannels 126 includes a buffer input orifice and a control valve and is configured to dispense a buffer to facilitate movement of the crosslinkable material within dispense channel 110.

[0022] In additional embodiments, the first and / or second shell channel inlets 104, 130 include two or more sub-channels configured to deliver fluid to the first and / or second shell channel outlets 106, 132, with each sub-channel converging at a separate fluid distribution orifice connecting the first and / or second shell channel inlets 104, 130 to the apex 116 of the upper curved surface 114 of the first and / or second shell channel outlets 106, 132. In preferred embodiments, the separate fluid distribution orifices are located on opposite sides of the first and / or second shell channel outlets 106, 132. In exemplary embodiments, the upper curved surface 114 has a parabolic or elliptical shape.

[0023] In one embodiment, the first shell channel includes at least one fluid distribution orifice connecting the first shell channel inlet 104 and the apex 116 of the upper curved surface 114 of the first shell channel outlet 106, such that fluid distributes along the upper curved surface 114 and around the first shell channel outlet 106; preferably, the upper curved surface 114 of the first shell channel outlet 106 has a parabolic or elliptical shape. In one embodiment, the first shell channel includes at least two first shell inlet sub-channels 126 that converge at or proximate to one fluid distribution orifice. In another embodiment, the first shell channel includes at least two first shell inlet sub-channels 126 that converge at or proximate to two fluid distribution orifices, preferably located on opposite sides of the first shell channel outlet 106.

[0024] In another embodiment, the second shell channel 128 includes at least one fluid distribution orifice connecting the second shell channel inlet 130 and the apex 116 of the upper curved surface 114 of the second shell channel outlet 132, such that the fluid distributes along the upper curved surface 114 and around the second shell channel outlet 132; preferably, the upper curved surface 114 of the second shell channel outlet 132 has a parabolic or elliptical shape. In one embodiment, the second shell channel 128 includes at least two second shell inlet sub-channels 126 that converge at or proximate to one fluid distribution orifice. In another embodiment, the second shell channel 128 includes at least two second shell inlet sub-channels 126 that converge at or proximate to two fluid distribution orifices, preferably located on opposite sides of the second shell channel outlet 132.

[0025] In some embodiments, the print head further includes a sheath flow channel 118 that converges with dispense channel 110 at a sheath fluid intersection disposed between the fluid focusing chamber(s) and the distal end of dispense channel 110. In some embodiments, sheath flow channel 118 comprises multiple sheath flow sub-channels that converge toward dispense channel 110 via sheath fluid chamber 120. In a preferred embodiment, sheath fluid chamber 120 has a frusto-conical shape configured to focus fluid toward dispense channel 110. In some embodiments, the sheath fluid intersection is located in the last / final downstream layer of the print head. In some embodiments, dispense channel 110 extends from the penultimate layer of the print head to sheath fluid chamber 120 in the last downstream layer.

[0026] In one embodiment, the minimum diameter of the frustum at the exit of the fluid focus chamber and the sheath fluid chamber 120 is the same and can be varied to adjust the overall fiber diameter, for example, between about 0.01 mm and about 5 mm. In some embodiments, the print head further includes a dispensing orifice disposed at the distal end of the dispensing channel 110. In some embodiments, the print head further includes an extension tip comprising a tube having an outer surface configured to fit within a portion of the dispensing channel 110 and an inner surface configured to align with the dispensing channel 110 (defining a hollow space within the tube).

[0027] In one embodiment, the sheath flow channel 118 includes a sheath fluid input orifice and a control valve, and preferably the print head is configured to dispense the sheath fluid through the sheath flow channel 118. In some embodiments, the sheath fluid includes a chemical crosslinker. In some embodiments, the sheath fluid includes an aqueous solvent.

[0028] In another aspect, the invention provides a print head including multiple stacked, preferably bonded, layers forming multiple fluid channels, including a core channel, multiple shell channels, and a fluid focus chamber converging toward dispense channel 110, wherein the core channel is in fluid communication with the fluid focus chamber and extends longitudinally through a central region of the fluid focus chamber in alignment with dispense channel 110; the multiple shell channels are concentrically arranged around the core channel in the same layer of the print head; the inner shell channel is in fluid communication with the fluid focus chamber and extends into the fluid focus chamber a longer length than the outer shell channels; the core channel extends into the fluid focus chamber a longer length than either shell channel; and sheath flow channel 118 converges with dispense channel 110 at a sheath fluid intersection located between the fluid focus chamber and the distal end of dispense channel 110.

[0029] In some embodiments, the print head further includes a plurality of fluid distribution orifices configured to dispense fluid around the plurality of shell channels, each fluid distribution orifice individually connecting a respective shell channel inlet 122 to an apex 116 of the upper curved surface 114 of a corresponding shell channel outlet of the plurality of shell channels, preferably the upper curved surface 114 of the second shell channel outlet 132 having a parabolic or elliptical shape. In an exemplary embodiment, at least one shell channel of the plurality of shell channels has a tapered width that increases with increasing longitudinal depth into the housing.

[0030] In some embodiments, the print head further includes third, fourth, fifth, and / or sixth shell channels having at least one inlet and outlet, each of the third, fourth, fifth, and / or sixth shell channel outlets being concentrically disposed around a previous shell channel outlet in a multi-channel enclosure in the same layer of the print head and in fluid communication with a fluid focus chamber. In some embodiments, the fluid focus chamber is disposed in a separate layer of the print head, and the shell channel outlets extend from a previous layer of the print head into a second fluid focus chamber in an adjacent, downstream layer. In a preferred embodiment, each of the third, fourth, fifth, and / or sixth shell channel outlets extends a shorter distance into the multi-channel enclosure than the previous shell channel outlet, and the core channel extends further into the multi-channel enclosure than the first shell channel.

[0031] In an alternative embodiment, the print head further includes third, fourth, fifth, and / or sixth shell channels having at least one inlet and outlet, and third, fourth, fifth, and / or sixth multichannel enclosures disposed between second fluid focusing chamber 136 and the distal end of dispense channel 110, the third, fourth, fifth, and / or sixth shell channel outlets, and the third, fourth, fifth, and / or sixth fluid focusing chambers, and dispense channel 110 is disposed at the center of each multichannel enclosure. a third, fourth, fifth, and / or sixth shell channel outlet disposed concentrically around dispense channel 110, fluidly communicating with the inlet of each fluid focusing chamber and extending a first vertical depth into the multichannel enclosure; a third, fourth, fifth, and / or sixth shell channel outlet disposed concentrically around dispense channel 110, fluidly communicating with the inlet of each fluid focusing chamber and extending a second vertical depth into the respective multichannel enclosure; the third, fourth, fifth, and / or sixth fluid focusing chambers converge toward dispense channel 110; preferably, the third, fourth, fifth, and / or sixth fluid focusing chambers comprise a frustoconical shape configured to focus fluid toward dispense channel 110. In some embodiments, the third, fourth, fifth, and / or sixth multichannel enclosures are disposed in successive layers of the print head. In some embodiments, the shell channel outlets can extend from a previous layer of the print head into the respective fluid focusing chambers of an adjacent, downstream layer of the print head.

[0032] In one preferred embodiment, the present invention discloses a printhead including a plurality of laminated layers forming multiple fluid channels, including: a core channel including at least two core inlet sub-channels with different fluid reservoirs, input orifices, and control valves that converge to form one core channel outlet 102 in fluid communication with a first fluid focusing chamber 112; a first shell channel including at least two shell inlet sub-channels 126 with different fluid reservoirs, input orifices, and control valves that converge to form one shell channel outlet in fluid communication with a second fluid focusing chamber 136; a dispense channel 110 with fluid focusing chambers converging toward the dispense channel, preferably comprising a conical frustum shape configured to focus fluid toward the dispense channel 110; and a sheath flow channel 118 that converges with dispense channel 110 at a sheath fluid intersection located between the second fluid focusing intersection and the distal end of dispense channel 110. In some embodiments, at least two core inlet subchannels converge at or proximal to the core channel outlet 102, preferably in the same or immediately preceding layer of the printhead as the core channel outlet 102. In some embodiments, the first shell channel includes three shell inlet subchannels 126, one of which is connected to a fluid reservoir containing a buffer solution.

[0033] In some embodiments, the core channel further comprises at least one fluid distribution orifice configured to distribute fluid around the core channel outlet 102, preferably the at least one fluid distribution orifice connects the convergent core channel inlet with the apex 116 of the upper curved surface 114 of the core channel outlet 102, and even more preferably the upper curved surface 114 has a parabolic or elliptical shape.

[0034] An aspect of the invention includes a system for generating a fiber structure, the system comprising a print head comprising a core channel having an inlet and an outlet, a first shell channel having an inlet and an outlet, a multi-channel enclosure, and a dispensing channel 110, the multi-channel enclosure including the core channel outlet 102, the first shell channel outlet 106, and a fluid focus chamber, the core channel outlet 102 being disposed in a central region of the multi-channel enclosure and in fluid communication with an inlet of the fluid focus chamber and extending a first vertical depth into the multi-channel enclosure, preferably the core channel outlet 102 extending a first vertical depth into the fluid focus chamber aligned with the dispensing channel 110, and the first shell channel outlet 106 being disposed in a central region of the multi-channel enclosure and in fluid communication with an inlet of the fluid focus chamber the print head, the print head being concentrically disposed around the nozzle, in fluid communication with the inlet of the fluid focusing chamber and extending a second vertical depth into the multichannel enclosure, the fluid focusing chamber converging toward the dispense channel 110, preferably the fluid focusing chamber comprising a frustoconical shape configured to focus fluid toward the dispense channel 110; a sheath flow channel 118 converging with the dispense channel 110 at a sheath fluid intersection located between a first fluid focusing intersection and the distal end of the dispense channel 110; a receiving surface for receiving a first layer of material dispensed from the print head; and a positioning component for positioning a dispensing orifice of the print head in 3D space, the positioning component being operably coupled to the print head.

[0035] In some embodiments, the system further comprises a programmable control processor for controlling the positioning components and for controlling the flow rate of one or more fluids through the print head. In some embodiments, the system further comprises a fluid removal component configured to remove excess fluid dispensed from the print head. In some embodiments, the fluid removal component comprises a porous membrane configured to allow the passage of excess fluid. In some embodiments, the fluid removal component comprises an absorbent material. In some embodiments, the fluid removal component comprises a vacuum configured to aspirate excess fluid. In some embodiments, the vacuum is applied below the receiving surface. In some embodiments, the vacuum is applied above the receiving surface. In some embodiments, the vacuum is applied via one or more vacuum channels on the print head. In some embodiments, the one or more vacuum channels are disposed near the dispensing orifices on the print head.

[0036] In some embodiments, the system further comprises a pressure control component configured to regulate the flow rate of one or more fluids through the print head. In some embodiments, the system further comprises one or more fluid reservoirs in fluid communication with the print head. In some embodiments, the fluid reservoir comprises a sheath fluid. In some embodiments, the sheath fluid comprises a cross-linking liquid configured to solidify the input material. In some embodiments, the cross-linking liquid comprises a divalent cation. In some embodiments, the divalent cation is Ca++. In some embodiments, the fluid reservoir comprises a buffer. In some embodiments, the buffer is miscible with the input material. In some embodiments, the fluid reservoir comprises the input material. In some embodiments, the input material comprises a cross-linkable material, such as a hydrogel. In some embodiments, the hydrogel comprises alginate. In some embodiments, the alginate is depolymerized alginate. In some embodiments, the input material comprises one or more living cells. In some embodiments, the input material comprises an extracellular matrix material. In some embodiments, the input material comprises an active agent.

[0037] In some embodiments, the system further comprises a print head comprising at least two shell inlet subchannels 126 connected to fluid reservoirs comprising different input materials, and / or a first shell channel comprising at least two shell subchannels, and the method comprises generating a core-shell fiber structure comprising a first input material and a second input material. In some embodiments, the method comprises dispensing the first and second input materials through the first and second shell channels to generate a solidified fiber structure comprising different concentric shells. In some embodiments, the method comprises dispensing the first and second input materials through the shell inlet subchannels 126 to generate a solidified fiber structure comprising different shell materials along the length of the continuous fiber structure.

[0038] In some embodiments, the print head is configured to generate a constant mass flow rate through the dispensing channel 110. In some embodiments, the system further comprises a bridging component. In some embodiments, the bridging component comprises a UV lamp. In some embodiments, the bridging component is disposed adjacent to the dispensing orifice.

[0039] Aspects of the invention include methods for producing solidified fiber structures, the method comprising: providing a system for producing a fiber structure, the system comprising a print head including a plurality of stacked, preferably bonded, layers forming a plurality of fluid channels, the plurality of fluid channels including a core channel having an inlet and an outlet, a first shell channel having an inlet and an outlet, a first multi-channel enclosure 108, and a dispensing channel 110, the multi-channel enclosure 108 including the core channel outlet 102, a first shell channel outlet 106, and a first fluid focus chamber 112, the core channel outlet 102 being disposed in a central region of the multi-channel enclosure 108 and in fluid communication with the inlet of the first fluid focus chamber 112 and extending a first vertical depth into the multi-channel enclosure 108, preferably the core channel outlet 102 extending a first vertical depth into the fluid focus chamber aligned with the dispensing channel 110, and the first shell channel outlet 106 being disposed concentrically around the core channel. the print head, the print head having a sheath flow channel 118, the sheath flow channel 118 converging with the dispense channel 110 at a sheath fluid intersection located between a first fluid focusing intersection and a distal end of the dispense channel 110; a receiving surface for receiving a first layer of material dispensed from the print head; a positioning component for positioning a dispensing orifice of the print head in 3D space, the positioning component being operably coupled to the print head; a programmable control processor for controlling the positioning component and for controlling the flow rate of one or more fluids through the print head; a first fluid reservoir containing a first input material; a second fluid reservoir containing a buffer solution; and a third fluid reservoir containing a sheath solution.the sheath solution comprises a cross-linking solution, the fluid reservoir being in fluid communication with a print head; passing a first input material through a dispensing channel; cross-linking the first input material with a cross-linking component to produce a solidified fiber structure; and dispensing the solidified fiber structure from a dispensing orifice of the print head;

[0040] In a preferred embodiment, the method includes simultaneously dispensing a buffer and / or sheath fluid through a core channel, one or more input materials through one or more shell channels, and a sheath fluid through a sheath flow channel 118 to form a hollow core within the printed fiber.

[0041] In some embodiments, the non-crosslinkable material in the core channel comprises a buffer solution, and the sheath fluid in the sheath flow channel 118 comprises a chemical crosslinker, and contact occurs at the sheath fluid intersection, solidifying the outer surface of the flow of crosslinkable material in the dispensing channel 110.

[0042] In some embodiments, the non-crosslinkable material in the core channel comprises a chemical crosslinker, the sheath fluid in the sheath flow channel 118 comprises an aqueous solvent, and contact occurs at the first fluid focusing intersection, causing the inner surface of the flow of crosslinkable material in the dispensing channel 110 to solidify.

[0043] In some embodiments, the non-crosslinkable material in the core channel comprises a chemical crosslinker, the sheath fluid in the sheath flow channel 118 comprises a chemical crosslinker, contact occurs at the first fluid focusing intersection and the inner surface of the flow of crosslinkable material solidifies in the dispensing channel, and contact occurs at the sheath fluid intersection and the outer surface of the flow of crosslinkable material solidifies in the dispensing channel 110.

[0044] In some embodiments, the method further includes encoding the planar structure to be printed into a programmable control processor and depositing a first layer of the solidified fiber structure onto the receiving surface to print the planar structure.

[0045] In some embodiments, the method further includes encoding the 3D structure to be printed into a programmable control processor and depositing a subsequent layer of the solidified fiber structure on top of the planar structure to print the 3D structure.

[0046] In another embodiment, the present invention provides a method for producing a continuously solidified fiber structure having a variable core and / or shell composition along the length of the fiber, the method comprising providing a system for producing a fiber structure, the system comprising: a print head including: a core channel including at least two core inlet sub-channels having separate fluid reservoirs, input orifices and control valves that converge to form one core channel outlet 102 in fluid communication with a first fluid focus chamber 112; a first shell channel including at least two shell inlet sub-channels 126 having separate fluid reservoirs, input orifices and control valves that converge to form one shell channel outlet in fluid communication with a second fluid focus chamber 136; and a dispense chamber 110, wherein the fluid focus chambers converge towards dispense channel 110, preferably the fluid focus chambers comprise a frusto-conical shape configured to focus fluid towards dispense channel 110; and a second a sheath flow channel 118 converging with dispense channel 110 at a sheath fluid intersection located between the fluid focusing intersection and the distal end of dispense channel 110; a receiving surface for receiving a first layer of material dispensed from the print head; a positioning component for positioning a dispense orifice of the print head in 3D space, said positioning component being operably coupled to the print head; a programmable control processor for controlling the positioning component and for controlling the flow rates of one or more fluids through the print head; a first fluid reservoir containing a first input material connected to the first core inlet subchannel; a second fluid reservoir containing a second input material connected to the second core inlet subchannel; a third fluid reservoir containing a third input material connected to the first shell inlet subchannel 126; a fourth fluid reservoir containing a fourth input material connected to the second shell inlet subchannel 126; and a fifth fluid reservoir containing a sheath solution connected to sheath flow channel 118;the sheath solution comprises a cross-linking solution, the fluid reservoir being in fluid communication with the print head; alternately passing first or second input materials through the dispensing channel 110 and simultaneously alternately passing third or fourth input materials through the dispensing channel 110; cross-linking the first, second, third and / or fourth input materials with a cross-linking component to produce a solidified fibrous structure; and dispensing the solidified fibrous structure from a dispensing orifice of the print head. In some embodiments, the first and / or second input materials comprise a non-cross-linkable material.

[0047] In another embodiment, the first shell channel includes three shell inlet subchannels, and a sixth fluid reservoir containing a buffer is connected to the third shell inlet subchannel 126, and the method includes passing the buffer through the dispensing channel 110 to move the crosslinkable material and terminate the fiber.

[0048] In a further aspect, bioprinted tissue fibers produced by the subject methods are also contemplated to have variable core and shell materials throughout the length of the fiber.

[0049] The present invention also solves the technical problem of creating a synthetic, perfusable, hollow tissue fiber that can attach to an external perfusion system without rupture, as detailed in Example 1 herein. In one aspect, the present invention provides a bioprinted tissue fiber comprising a lumen, a continuous inner shell layer surrounding the lumen, the continuous inner shell layer comprising a reinforcing hydrogel material at the distal and proximal ends of the fiber, and a biocompatible hydrogel material therebetween. The biocompatible hydrogel material preferably comprises at least one biological material, e.g., living cells, while the reinforcing hydrogel material is acellular. In another embodiment, the fiber further comprises a second, outer, continuous shell layer comprising a reinforcing hydrogel material.

[0050] In some embodiments, the reinforced hydrogel material is selected from, for example, alginate, chitosan, acrylated PEG, including, but not limited to, PEGDA, PEGTA, polyvinyl alcohol (PVA), PCL, and PLGA. In some embodiments, the biocompatible hydrogel material is selected from, for example, alginate, chitosan, acrylated PEG, collagen, laminin, fibronectin, vitronectin, fibrin / fibrinogen, decellularized tissue ECM, hyaluronic acid, gelatin, and ECM factors, including methacrylated gelatin. In exemplary embodiments, the reinforced hydrogel material comprises a higher concentration of alginate material, e.g., 3.5-4.5 wt%, preferably 3.8-4.2 wt%, and more preferably about 4 wt%, while the biocompatible hydrogel material comprises a lower concentration of alginate material, e.g., 1.0-1.5 wt%, preferably 1.2-1.4 wt%, and more preferably about 1.3 wt%.

[0051] In some embodiments, the at least one biological material comprises living cells, e.g., cells from endocrine and exocrine glands including the pancreas (alpha, beta, delta, epsilon, gamma), liver (hepatocytes, Kupffer, stellate, sinusoidal cells), thyroid gland (follicular cells), pineal gland (pineal cells), pituitary gland (somatotrophs, lactotrophs, gonadotrophs, corticotrophs, and thyrotrophs), thymus (thymocytes, thymic epithelial cells, thymic stromal cells), adrenal gland (cortical cells, chromaffin cells), ovary (granulosa cells), testis (Leydig cells), and gastrointestinal tract (enteroendocrine cells—gut, stomach, pancreas). In preferred embodiments, the at least one biological material comprises a cell population that expresses / secretes a biologically active agent, e.g., insulin, glucagon, ghrelin, pancreatic polypeptide, angiogenic factors, growth factors, hormones, antibodies, enzymes, proteins, exosomes, etc.

[0052] Embodiments of the present invention also include a method for producing perfusable hollow tissue fibers, the method comprising: providing a printhead according to the subject invention with a first shell channel including at least two shell inlet subchannels 126 with separate fluid reservoirs, input orifices, and control valves; dispensing a sheath fluid through the core channel, a reinforced hydrogel material through the first shell inlet subchannel 126, a biocompatible hydrogel material including one or more biomaterials through the second shell inlet subchannel 126, and a sheath fluid through the sheath flow channel 118; and transitioning between the reinforced hydrogel material and the biocompatible hydrogel material along the length of the printed fiber. In this method, reinforced materials can be incorporated into the ends of the perfusable fibers to enable attachment to an external perfusion system, such as via needle insertion. In another embodiment, the print head further includes a second shell channel 128 as described herein, and the method includes dispensing the same or a different reinforcing hydrogel material through the second shell channel 128 to create a concentric second shell around the first shell material to further strengthen the fiber and prevent rupture along the entire length of the fiber.

[0053] The present invention also solves the technical problem of creating a synthetic, perfusable, hollow tissue fiber that can attach to an external perfusion system without rupture, as detailed in Example 1 herein. In one aspect, the present invention provides a bioprinted tissue fiber comprising a lumen, a continuous inner shell layer surrounding the lumen, the continuous inner shell layer comprising a reinforcing hydrogel material at the distal and proximal ends of the fiber, and a biocompatible hydrogel material therebetween. The biocompatible hydrogel material preferably comprises at least one biological material, e.g., living cells, while the reinforcing hydrogel material is acellular. In another embodiment, the fiber further comprises a second, outer, continuous shell layer comprising a reinforcing hydrogel material. [Brief explanation of the drawings]

[0054] [Figure 1] FIG. 1 is a diagram of a concentric shell printhead design of the subject invention having one fluid dispensing orifice. [Figure 2] FIG. 1B is a side view and enlarged detail of the multi-channel enclosure and sheath flow chamber in the concentric shell printhead design of the subject invention having one fluid distribution orifice. [Figure 3] 1 illustrates and identifies the main components of the microfluidic pathway in the concentric shell printhead design of the subject invention, having one fluid dispensing orifice. [Figure 4] FIG. 1A is a diagram of the flow pattern through one fluid distribution orifice in the concentric shell printhead design of the subject invention; FIG. 1B is a diagram of the flow pattern through one fluid distribution orifice in the multi-channel enclosure and sheath flow chamber of the concentric shell printhead design of the present invention. [Figure 5] 1A-1C are side and enlarged detail views of the multi-channel enclosure and sheath flow chamber in a multi-shell concentric printhead design of the subject invention having two fluid input orifices. [Figure 6] 1 illustrates and identifies the main components of the microfluidic pathway in the multi-shell concentric printhead design of the subject invention having two fluid dispensing orifices. [Figure 7] FIG. 1A is a diagram of the flow pattern through two fluid distribution orifices in a multi-shell concentric printhead design of the subject invention having two fluid distribution orifices; FIG. 1B is a diagram of the flow pattern through two fluid distribution orifices in a multi-channel enclosure and sheath flow chamber of a multi-shell concentric printhead design of the present invention. [Figure 8A] 1 provides a diagram and enlarged detail view of an embodiment of the subject invention that includes two separate shell inlet sub-channels that converge into one fluid distribution orifice. [Figure 8B] 1 provides a diagram and enlarged detail view of an embodiment of the subject invention that includes two separate shell inlet sub-channels that converge into one fluid distribution orifice. [Figure 9A]1 provides a perspective view (A) of a printhead design in accordance with the subject invention, which includes three separate shell inlet sub-channels converging into one fluid dispensing orifice. [Figure 9B] FIG. 1B provides a top view of a printhead design in accordance with the subject invention, including three separate shell inlet sub-channels converging into one fluid dispensing orifice. [Figure 9C] 1 provides an exploded layer view (C) of a printhead design in accordance with the subject invention, including three separate shell inlet sub-channels converging into one fluid dispensing orifice. [Figure 10A] FIG. 1 provides a perspective view (A) of a printhead design according to the subject invention, including a second shell channel, a second multi-channel enclosure, and a second fluid focusing chamber in a different layer than the first fluid focusing chamber of the printhead. [Figure 10B] FIG. 1B provides a top view of a printhead design in accordance with the subject invention, including a second shell channel, a second multi-channel enclosure, and a second fluid focusing chamber in a different layer than the first fluid focusing chamber of the printhead. [Figure 10C] 1 provides an exploded layer view (C) of a printhead design according to the subject invention, including a second shell channel, a second multi-channel enclosure, and a second fluid focusing chamber on a different layer than the first fluid focusing chamber of the printhead. [Figure 11] 1 provides a perspective view of a printhead design in accordance with the subject invention, including a second shell channel, a second multi-channel enclosure, and a second fluid focusing chamber overlapping a first fluid focusing chamber, the second multi-channel enclosure overlapping the first fluid focusing chamber in the same layer of the printhead. [Figure 12] 1 provides various views of a perfusable hollow tissue fiber according to the subject invention. [Figure 13] 10 provides additional views of perfusable hollow tissue fibers according to the subject invention. [Figure 14]Various views of synthetic tissue fibers according to the subject invention are provided, including variable core and / or shell compositions along the length of the fiber. [Figure 15] Synthetic hollow tissue fibers according to the subject invention are provided having a range of lumen and fiber diameters. DETAILED DESCRIPTION OF THE INVENTION

[0055] Aspects of the invention include systems and methods for fabricating core-shell fiber structures, including hollow core fibers and multi-shell fibers, and for fabricating three-dimensional (3D) structures from digital files. In some embodiments, the printed fibers comprise living cells.

[0056] definition For purposes of interpreting this specification, the following definitions shall apply, except that where appropriate, terms used in the singular shall include the plural and vice versa. In the event that any definition set forth conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0057] As used herein, the term "displacement" refers to the ability of a first material or fluid to displace a second material or fluid from a given location. For example, in some embodiments, the buffer is configured to displace an input material from a location within dispense channel 110 (e.g., from the proximal end of dispense channel 110). In some embodiments, the displacement is instantaneous, occurring in less than about 1 second, e.g., about 900, 800, 700, 600, 500, 400, 300, 200, or 100 milliseconds or less.

[0058] As used herein, the term "miscibility" refers to the ability of two different liquids to form a homogeneous mixture when combined.

[0059] As used herein, the term "mass flow rate" refers to the mass of material passing through a given location per unit time. As used herein, the term "constant mass flow rate" refers to a mass flow rate that remains constant per unit time.

[0060] As used herein, the term "solidified" refers to a solid or semi-solid state of a material that maintains its shape fidelity and structural integrity upon deposition. As used herein, the term "shape fidelity" refers to the ability of a material to maintain its three-dimensional shape without significant spreading. In some embodiments, a solidified material has the ability to maintain its three-dimensional shape for a period of about 30 seconds or more, e.g., about 1, 10, or 30 minutes or more, e.g., about 1, 10, 24, or 48 hours or more. As used herein, the term "structural integrity" refers to the ability of a material to remain cohesive under load, including its own weight, while resisting fracture or flexing.

[0061] In some embodiments, the solidifying composition has a modulus of elasticity greater than about 5, 10, 15, 20, or 25 kilopascals (kPa), more preferably greater than about 30, 40, 50, 60, 70, 80, or 90 kPa, and even more preferably greater than about 100, 110, 120, or 130 kPa. Preferred modulus ranges include from about 5, 10, 15, 20, 25, or 50 Pa to about 80, 100, 120, or 140 kPa. In accordance with the subject invention, the modulus of elasticity of the input material can be advantageously varied depending on the intended function of the input material. In some embodiments, a lower modulus is used to support cell growth and migration, while in other embodiments, a much higher modulus may be used.

[0062] As used herein, the term "natural alginate polymer" refers to an alginate polymer that has been isolated and purified from one or more natural sources (eg, one or more species of brown seaweed or seaweed).

[0063] As used herein, the term "depolymerization" refers to the breaking down of polymer chains into monomers or other smaller units.

[0064] As used herein, the term "hydrogel" refers to a composition that contains water and a network or lattice of polymer chains that are hydrophilic.

[0065] As used herein, the term "sheath fluid" or "sheath liquid" refers to a fluid that is used at least in part to encase or "sheath" a material as it passes through a fluid channel. In some embodiments, the sheath fluid comprises an aqueous solvent, such as water or glycerol. In some embodiments, the sheath fluid comprises a chemical crosslinker. Non-limiting examples of crosslinkers include divalent cations (e.g., Ca 2+ , Ba 2+ , Sr 2+ ), thrombin, and pH-modifying chemicals such as baking soda.

[0066] As used herein, the term "excess sheath fluid" refers to a portion of the sheath fluid dispensed from a dispensing orifice that does not form part of the fiber structure printed using one or more embodiments of the systems or methods provided herein. For example, the excess sheath fluid may be useful for lubricating the passage of a material (e.g., a hydrogel) through the dispensing orifice through the dispensing channel 110 of the print head. When dispensed from the dispensing orifice, the excess sheath fluid may flow off the surface of the layer of dispensed material onto the receiving surface and may collect, i.e., pool, on the receiving surface.

[0067] As used herein, the term "channel length" refers to the linear distance traveled when tracing a fluid channel from a first location to a second location.

[0068] As used herein, the term "convergence angle" refers to the angle formed between two converging fluid channels. print head

[0069] Aspects of the present invention include print heads that can be used to fabricate one or more core-shell fiber structures, including multishell fibers and / or hollow fibers. Print heads according to embodiments of the present invention include multiple laminated layers, preferably bonded together to form a common housing or enclosure, forming multiple interconnected fluid channels that flow vertically within the layers, and are configured to fabricate core-shell fiber structures containing one or more input materials. In some embodiments, the print head is configured to fabricate solidified hollow fiber structures. In some embodiments, the print head is configured to fabricate solidified hollow fiber structures containing living cells.

[0070] In some embodiments, the print head includes a dispensing channel 110 having a distal end and a proximal end. Dispensing channels according to embodiments of the invention may have a channel length ranging from about 1 mm to about 100 mm, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or about 95 mm. Dispensing channels according to embodiments of the invention may have a width or diameter ranging from about 10 μm to about 5 mm, such as about 25, 50, 75, or 100 μm, or a width or diameter of about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, or 3.0 mm. Dispensing channels according to embodiments of the invention may have depths ranging from about 10 μm to about 5 mm, such as about 25, 50, 75, or 100 μm, or for example, about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, or 3.0 mm. Dispensing channels according to embodiments of the invention may have any suitable cross-sectional shape, such as a circular, oval, square, or rectangular cross-sectional shape.

[0071] In some embodiments, dispensing channel 110 includes a dispensing orifice. In some embodiments, the dispensing orifice is located at the distal end of dispensing channel 110. Dispensing orifices according to embodiments of the invention can have diameters ranging from about 10 μm to about 5 mm, such as about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 μm, or, for example, about 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or 950 μm. Dispensing orifices according to embodiments of the invention can have any suitable cross-sectional shape, such as a circular, oval, square, or rectangular cross-sectional shape.

[0072] In some embodiments, the print head further comprises an extended tip having an orifice for dispensing material from the print head. Such an extended tip facilitates precise dispensing and deposition of material in a confined area, such as a well in a multiwell plate (e.g., a standard microtiter plate, multiwell plate, or microplate having 6, 24, or 96 or more wells) or a Petri dish. In some embodiments, the extended tip comprises a tube (e.g., made of plastic, glass, or metal) having an exterior configured to fit within a portion of the dispensing channel 110 and an interior surface configured to align with the dispensing channel (defining a hollow space within the tube). The extended tip can be inserted into the dispensing channel 110, thereby extending the length of the dispensing channel and facilitating deposition of material dispensed from the orifice in the extended tip into a confined space, such as a well plate insert or Petri dish.

[0073] Printheads according to embodiments of the present invention include one or more core channels. In certain embodiments, the one or more core channels converge with dispense channel 110 at a proximal end of dispense channel 110. In some embodiments, the core channels converge with dispense channel 110 at a convergence angle ranging from about 0 to 180 degrees, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, or 175 degrees. Core channels according to embodiments of the present invention may have any suitable channel length. In some embodiments, the core channel has a channel length ranging from about 100 μm to about 100 mm, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 mm. Core channels according to embodiments of the invention can have a width or diameter ranging from about 10 μm to about 5 mm, e.g., about 25, 50, 75, or 100 μm, or e.g., about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, or 3.0 mm. Material channels according to embodiments of the invention may have a depth ranging from about 10 μm to about 5 mm, such as about 25, 50, 75, or 100 μm, or for example, about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, or 3.0 mm.

[0074] In some embodiments, the print head includes at least two core subchannels with the same or different fluid reservoirs, input orifices, and control valves that converge to form a single core channel outlet 102 in fluid communication with the fluid focus chamber. In preferred embodiments, the at least two core subchannels converge at or proximate to the core channel outlet 102, multichannel enclosure 108, and / or fluid distribution orifices described further herein, e.g., within about 100 μm to about 50 mm, to shorten travel distances and move the material transition point closer to the freezing point. The inventors have determined that this prevents smearing between material transitions within the printed fabric. In particularly preferred embodiments, the at least two subchannels converge at a layer immediately preceding the print head or at the same layer of the print head as the core channel outlet 102, multichannel enclosure, and / or fluid distribution orifice. In some embodiments, the channel length between the location where the subchannels converge and the core channel outlet 102, multichannel enclosure 108, and / or fluid dispensing orifice ranges from about 100 μm to about 50 mm, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mm. In some embodiments, the print head comprises a plurality of core subchannels ranging from 3 to 10, e.g., 4, 5, 6, 7, 8, or 9 subchannels. Core channels according to embodiments of the invention can have any suitable cross-sectional shape, e.g., circular, oval, square, or rectangular. In some embodiments, the print head is configured to dispense a non-cross-linkable material through the core channel(s).

[0075] A printhead according to an embodiment of the invention includes a core channel having an inlet and an outlet, a first shell channel having an inlet and an outlet, a multi-channel enclosure 108, and a dispensing channel 110, the multi-channel enclosure 108 including the core channel outlet 102, the first shell channel outlet 106, and a fluid focus chamber, the core channel outlet 102 being disposed in a central region of the multi-channel enclosure 108 and in fluid communication with an inlet of the fluid focus chamber, the multi-channel enclosure 108 having a first vertical depth. Preferably, core channel outlet 102 extends a first vertical depth into fluid focusing chamber 106 aligned with dispense channel 110, and first shell channel outlet 106 is concentrically disposed around the core channel and in fluid communication with the inlet of the fluid focusing chamber, extending a second vertical depth into multichannel enclosure 108, the fluid focusing chamber converging toward dispense channel 110, and preferably, the fluid focusing chamber comprises a frustoconical shape configured to focus fluid toward dispense channel 110. In some embodiments, first shell channel outlet 106 has a gradient width that increases with increasing depth into multichannel enclosure 108. In an exemplary embodiment, first shell channel outlet 106 comprises a hollow cylinder having an axis of rotation that does not intersect with core channel outlet 102.

[0076] In some embodiments, the core channel outlet 102 extends through a majority of the length of the multichannel enclosure 108. In preferred embodiments, the first vertical depth is greater than the second vertical depth, such that the core channel outlet 102 extends further into the multichannel enclosure 108 and / or the first fluid focusing chamber 112 than the first shell channel outlet 106. In alternative embodiments, the second vertical depth is greater than the first vertical depth, such that the first shell channel outlet 106 extends further into the multichannel enclosure 108 than the core channel outlet 102.

[0077] In some embodiments, the print head further includes a second shell channel 128 having an inlet and an outlet, the second shell channel outlet 132 and / or inlet 130 being concentrically disposed around the first shell channel outlet 106 of the multi-channel enclosure 108 in the same layer of the print head and in fluid communication with the fluid focus chamber. In a preferred embodiment, the first shell channel outlet 106 extends further into the multi-channel enclosure 108 than the second shell channel outlet 132. In some embodiments, the second shell channel inlet 130 may be adjacent to the first shell channel outlet 106 in the same layer of the print head and in fluid communication with the first fluid focus chamber 112.

[0078] In an alternative embodiment, the print head further includes a second shell channel having at least one inlet and outlet, and a second multichannel enclosure disposed between the first fluid focusing chamber and the distal end of the dispense channel, the second multichannel enclosure comprising the dispense channel, the second shell channel outlet, and a second fluid focusing chamber, the dispense channel disposed in a central region of the second multichannel enclosure, in fluid communication with the inlet of the second fluid focusing chamber, and extending a first vertical depth into the multichannel enclosure, preferably the second shell channel outlet disposed concentrically around the dispense channel, in fluid communication with the inlet of the second fluid focusing chamber, and extending a second vertical depth into the multichannel enclosure, the second fluid focusing chamber converging towards the dispense channel, preferably the second fluid focusing chamber comprising a frustoconical shape configured to focus fluid towards the dispense channel. In some embodiments, the first and second multichannel enclosures, or portions thereof, are disposed in the same layer of the print head, and the second multichannel enclosure may overlap the first fluid focusing chamber, as shown, for example, in Figure 11. In some embodiments, the first and second multichannel enclosures are disposed in successive layers of the print head, as shown, for example, in Figure 10A. In some embodiments, the second fluid focusing chamber is disposed in a separate layer of the print head, and the second shell channel outlet extends from a previous layer of the print head into the second fluid focusing chamber of an adjacent, downstream layer.

[0079] Core and shell channels according to embodiments of the invention can have any suitable length. In some embodiments, the core or shell channels have a channel length ranging from about 100 μm to about 100 mm, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 mm. Core and shell channels according to embodiments of the invention can have a width or diameter ranging from about 10 μm to about 5 mm, e.g., about 25, 50, 75, or 100 μm, or a width or diameter of about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, or 3.0 mm. Core and shell channels according to embodiments of the invention can have depths ranging from about 10 μm to about 5 mm, such as about 25, 50, 75, or 100 μm, or for example, about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, or 3.0 mm. Core and shell channels according to embodiments of the invention can have any suitable cross-sectional shape, such as circular, oval, square, or rectangular cross-sectional shapes.

[0080] In some embodiments, the first shell channel, the second shell channel 128, or both, further comprise at least one fluid distribution orifice configured to distribute fluid around the first shell channel outlet 106 and / or the second shell channel outlet 132. Preferably, the fluid distribution orifice connects the first and / or second shell channel inlet 104, 130 to the apex 116 of the upper curved surface 114 of the first and / or second shell channel outlet 106, 132. In some embodiments, the first and / or second shell channel includes at least two shell sub-channels, which may converge to one fluid distribution orifice or may lead to separate fluid distribution orifices. The shell sub-channels may be in fluid communication with the same fluid reservoir, input orifice, and control valve, or may be in fluid communication with separate fluid reservoirs, input orifices, and control valves. In some embodiments, the print head comprises a plurality of shell subchannels ranging from 3 to 10, e.g., 4, 5, 6, 7, 8, or 9. Shell channels and shell subchannels according to embodiments of the invention can have any suitable cross-sectional shape, e.g., circular, oval, square, or rectangular.

[0081] In additional embodiments, the first and / or second shell channel inlets 104, 130 include two or more sub-channels configured to deliver fluid to the first and / or second shell channel outlets 106, 132, each sub-channel including a separate fluid distribution orifice connecting the first and / or second shell channel inlets 104, 130 to the apex 116 of the upper curved surface 114 of the first and / or second shell channel outlets 106, 132, preferably the upper curved surface 114 of the first and / or second shell channel outlets 106, 132 has a parabolic or elliptical shape. In preferred embodiments, the separate fluid input orifices are located on opposite sides of the first and / or second shell channel outlets 106, 132. In exemplary embodiments, the upper curved surface 114 has a parabolic or elliptical shape.

[0082] In one embodiment, the first shell channel includes at least one fluid distribution orifice connecting the first shell channel inlet 104 and the apex 116 of the curved upper surface 114 of the first shell channel outlet 106, such that fluid distributes along the curved upper surface 114 and around the first shell channel outlet 106. In a further embodiment, the first shell channel includes two fluid distribution orifices located on opposite sides of the first shell channel outlet 106. In another embodiment, the second shell channel 128 includes at least one fluid distribution orifice connecting the second shell channel inlet 130 and the apex 116 of the curved upper surface 114 of the second shell channel outlet 132, such that fluid distributes along the curved upper surface 114 and around the second shell channel outlet 132. In a further embodiment, the second shell channel 128 includes two fluid distribution orifices located on opposite sides of the second shell channel outlet 132.

[0083] Printheads according to embodiments of the present invention include a sheath flow channel 118. In certain embodiments, the sheath flow channel 118 converges with the dispense channel 110 at a sheath fluid intersection located between the first fluid focusing intersection and the distal end of the dispense channel 110. In some embodiments, the sheath flow channel 118 converges with the dispense channel 110 at a convergence angle in the range of about 0 to 180 degrees, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, or 175 degrees. In some embodiments, the distance between the proximal end of dispense channel 110 and the sheath fluid intersection ranges from about 10 μm to about 100 mm, such as about 25, 50, 75, or 100 μm, or for example, about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 mm. In some embodiments, the distance between the distal end of dispense channel 110 and the sheath fluid intersection ranges from about 10 μm to about 100 mm, such as about 25, 50, 75, or 100 μm, or for example, about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 mm.

[0084] Sheath flow channels according to embodiments of the invention can have any suitable length. In some embodiments, sheath flow channel 118 has a channel length ranging from about 100 μm to about 100 mm, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 mm. Sheath flow channels according to embodiments of the invention can have a width or diameter ranging from about 10 μm to about 5 mm, e.g., about 25, 50, 75, or 100 μm, or a width or diameter of about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, or 3.0 mm. Sheath flow channels according to embodiments of the present invention can have depths ranging from about 10 μm to about 5 mm, such as about 25, 50, 75, or 100 μm, or about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, or 3.0 mm. In some embodiments, sheath flow channel 118 comprises two or more sheath flow subchannels. In some embodiments, sheath flow channel 118 branches into a plurality of sheath flow subchannels ranging from 3 to 10, such as 4, 5, 6, 7, 8, or 9. In some embodiments, two or more sheath flow subchannels converge with dispense channel 110 at a sheath fluid intersection. Sheath flow channels according to embodiments of the present invention can have any suitable cross-sectional shape, such as a circular, oval, square, or rectangular cross-sectional shape.

[0085] Fluid channels according to embodiments of the invention generally include one or more input orifices through which fluid can be introduced into the channel, which are generally located in the first or top layer of the printhead stack. In some embodiments, the fluid channels include control valves configured to regulate fluid flow through the fluid channels, which are generally located in the second top layer of the printhead stack. In some embodiments, the channel length between the input orifices and the control valves ranges from about 100 μm to about 100 mm, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 mm. In some embodiments, the channel length between the control valve and the point where the channel converges with dispense channel 110 ranges from about 100 μm to about 100 mm, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 mm.

[0086] Print heads according to embodiments of the present invention can be made from any suitable material, including, but not limited to, plastic (e.g., polymeric materials), glass, metal, ceramic, or any combination thereof. In a preferred embodiment, the print head is fabricated using known microfluidic molding techniques (e.g., casting, imprinting, or injection molding) and one or more moldable polymers, such as polydimethylsiloxane (PDMS), polycarbonate (PC), cyclic olefin polymer (COP), polyethylene terephthalate (PET), polyethylene (PE), high-density polyethylene (HDPE), and polystyrene (PS). Suitable bonding processes include solvent bonding, plasma bonding, adhesive bonding, ultrasonic bonding, and vulcanization. Alternatively, commercially available 3D printing technologies can be used to fabricate the print head.

[0087] In some embodiments, the print head comprises a material that is at least partially transparent to light (e.g., ultraviolet (UV) light). In some embodiments, the print head is made entirely of a transparent material. In certain embodiments, the portion of the print head surrounding or directly adjacent to the dispense channel 110 comprises a material that is partially or completely light transmissive. Such print heads can be used in conjunction with input materials configured to be crosslinked by light energy (e.g., photocrosslinkable input materials).

[0088] Aspects of the present invention include a light module configured to expose a photo-crosslinkable dosing material to electromagnetic radiation to crosslink the dosing material. Light modules according to embodiments of the present invention may be integrated into a print head or may be separate components of a printing system. In some embodiments, the light module exposes the dosing material while it is present in dispensing channel 110. In some embodiments, the light module exposes the dosing material after it is dispensed from dispensing channel 110. In some embodiments, a print head includes multiple light modules, where a first light module is configured to expose the dosing material while it is present in dispensing channel 110 and a second light module is configured to expose the dosing material after it is dispensed from dispensing channel 110.

[0089] In some embodiments, the light module is adjustable with respect to wavelength, intensity, exposure time, or any combination thereof. In some embodiments, the light module includes one or more optionally engaged attenuating filters that, when engaged, adjust the light intensity. In some embodiments, the light module is configured to emit UV light, and the wavelength of the light emitted from the module ranges from about 10 nm to about 400 nm, e.g., about 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, or 375 nm. In some embodiments, suitable UV light sources include, by way of non-limiting example, a UV lamp, a UV fluorescent lamp, a UV LED, a UV laser, or any combination thereof.

[0090] As outlined above, aspects of the invention include a print head comprising a dispense channel 110, where one or more material channels and optionally a buffer channel converge at the proximal end of the dispense channel 110. The subject print head is configured to dispense buffer and / or sheath fluid simultaneously with one or more cross-linkable materials to form hollow cores in printed fibers. In some embodiments, the print head is configured to maintain a constant mass flow rate through the dispense channel 110. In this manner, the subject print head is configured to promote smooth and continuous flow of one or more input materials (or a mixture of one or more input materials) and buffer and / or sheath fluid through the dispense channel 110.

[0091] As outlined above, additional aspects of the present invention include print heads with dispense channel 110, where one or more sheath flow channels 118 converge with dispense channel 110 at a sheath fluid intersection located between a first fluid focusing intersection and the distal end of dispense channel 110. During use of the subject print heads, input material flowing through dispense channel 110 can be cross-linked both internally by sheath fluid flowing through the core channel and externally by sheath fluid flowing through sheath flow channels 118.

[0092] In a preferred embodiment, the present invention provides a print head including multiple stacked, preferably bonded, layers forming multiple fluid channels, including a core channel, multiple shell channels, and a fluid focus chamber converging toward dispense channel 110, wherein the core channel is in fluid communication with the fluid focus chamber and extends longitudinally in alignment with dispense channel 110 through a central region of the fluid focus chamber; the multiple shell channels are concentrically arranged around the core channel in the same layer of the print head; the inner shell channel is in fluid communication with the fluid focus chamber and extends into the fluid focus chamber a longer length than the outer shell channels; the core channel extends into the fluid focus chamber a longer length than either shell channel; and sheath flow channel 118 converges with dispense channel 110 at a sheath fluid intersection located between the fluid focus chamber and the distal end of dispense channel 110.

[0093] In some embodiments, the print head further includes a plurality of fluid distribution orifices configured to distribute fluid around the plurality of shell channels, each fluid distribution orifice individually connecting a respective shell channel inlet 122 to an apex 116 of the upper curved surface 114 of a corresponding shell channel outlet of the plurality of shell channels, preferably the upper curved surface 114 of the shell channel outlet having a parabolic or elliptical shape. In an exemplary embodiment, at least one shell channel of the plurality of shell channels has a tapered width that increases with increasing longitudinal depth into the housing.

[0094] In some embodiments, the print head further includes a third, fourth, fifth, and / or sixth shell channel having an inlet and an outlet, each of the third, fourth, fifth, and / or sixth shell channel outlets being concentrically disposed about the immediately preceding shell channel outlet within the multi-channel enclosure and in fluid communication with the fluid focusing chamber. In preferred embodiments, each of the third, fourth, fifth, and / or sixth shell channel outlets extends a shorter distance into the multi-channel enclosure than the immediately preceding shell channel outlet, and the core channel extends further into the multi-channel enclosure than the first shell channel.

[0095] In another preferred embodiment, the present invention provides a core channel including at least two core inlet sub-channels with separate fluid reservoirs, input orifices, and control valves that converge to form one core channel outlet 102 in fluid communication with a first fluid focus chamber 112, preferably wherein said at least two core inlet sub-channels converge at or proximal to said core channel outlet 102; and a shell channel with separate fluid reservoirs, input orifices, and control valves that converge to form one shell channel outlet in fluid communication with a second fluid focus chamber 136. The printhead includes a plurality of stacked layers forming a plurality of fluid channels, the stacked layers comprising: a first shell channel including at least two shell inlet sub-channels 126 having control valves; a dispense channel 110 having fluid focusing chambers converging toward the dispense channel 110, preferably including a conical frustum shape configured to focus fluid toward the dispense channel 110; and a sheath flow channel 118 converging with the dispense channel 110 at a sheath fluid intersection located between the second fluid focusing intersection and the distal end of the dispense channel 110. In a further embodiment, the first shell channel includes three shell inlet sub-channels 126, one of which is connected to a fluid reservoir containing a buffer solution. In some embodiments, the core channel further comprises at least one fluid distribution orifice configured to distribute fluid around the core channel outlet 102, preferably the at least one fluid distribution orifice connects the convergent core channel inlet with the apex 116 of the upper curved surface 114 of the core channel outlet 102, and even more preferably the upper curved surface 114 has a parabolic or elliptical shape.

[0096] Printing System: Aspects of the present invention include printing systems and associated components configured to operate in conjunction with the subject printheads to perform the subject methods. In some embodiments, the printing system comprises a single printhead described herein. In some embodiments, the printing system comprises multiple printheads, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 individual printheads described herein. In some embodiments, the printheads are fluidly isolated from the printing system, such that all fluids involved in the printing process remain isolated within the printhead and only contact the receiving surface of the printing system during the printing process (described below). In some embodiments, the printheads are configured to be operably connected to the printing system without fluids involved in the printing process contacting components of the printing system. In some embodiments, one or more printheads can be removed from and / or added to the printing system before, during, and / or after the printing process. Thus, in some embodiments, the subject printheads are modular components of the subject printing systems.

[0097] In some embodiments, the printing system includes a receiving surface upon which a first layer of material dispensed from a dispensing orifice of a print head is deposited. In some embodiments, the receiving surface comprises a solid material. In some embodiments, the receiving surface comprises a porous material. For example, in some embodiments, the porous material has sufficient porosity to allow fluid to pass through the porous material. In some embodiments, the receiving surface is generally planar, thereby providing a flat surface upon which the first layer of dispensed material can be deposited. In some embodiments, the receiving surface has a shape corresponding to the three-dimensional structure to be printed, thereby facilitating printing of three-dimensional structures having a non-planar first layer.

[0098] In some embodiments, the receiving surface comprises a vacuum component configured to apply suction to the receiving surface from one or more vacuum sources. In some embodiments, the receiving surface comprises one or more vacuum channels configured to apply suction to the receiving surface. In some embodiments, the receiving surface with the vacuum component is configured to suction excess fluid from the receiving surface before, during, and / or after performing a printing process.

[0099] In some embodiments, the receiving surface is a non-cytotoxic surface onto which the printing system dispenses one or more fibrous structures. In some embodiments, the printing system includes a printer stage. In some embodiments, the receiving surface is a surface of the printer stage. In some embodiments, the receiving surface is a component separate from the printer stage but attached to or supported by the printer stage. In some embodiments, the receiving surface is flat or substantially flat. In some embodiments, the receiving surface is smooth or substantially smooth. In some embodiments, the receiving surface is substantially flat and substantially smooth. In some embodiments, the receiving surface is configured to correspond to the shape, size, texture, or geometry of the structures to be printed. In some embodiments, the receiving surface controls or influences the size, shape, texture, or geometry of the structures to be printed.

[0100] In some embodiments, the receiving surface comprises one or more modular components that are configured to be operably connected to the printing system but are separable from the printing system. In some embodiments, the receiving surface is a disposable receiving surface. In some embodiments, the receiving surface is configured for sterilization. In some embodiments, the entire fluid path of the printing system is disposable, meaning that all components of the printing system that come into contact with one or more fluids involved in the printing process are disposable and can be removed from the printing system and replaced with clean components.

[0101] In some embodiments, the receiving surface is configured to be operably coupled to one or more different receiving vessels. For example, in some embodiments, the receiving surface has a circular portion sized to be operably coupled to a circular receiving vessel (e.g., a Petri dish). In some embodiments, the receiving surface has a square or rectangular portion sized to be operably coupled to a square or rectangular receiving vessel (e.g., a multiwell plate (e.g., a 6-well plate)). Receiving surfaces according to embodiments of the present invention can have any suitable size or geometric form to accommodate a suitable receiving vessel.

[0102] In some embodiments, the printing system includes a temperature regulation component configured to regulate the temperature of the receiving surface. In some embodiments, the temperature regulation component regulates and / or maintains the temperature of the receiving surface at ambient temperature. In some embodiments, the temperature regulation component regulates and / or maintains the temperature of the print head, printer stage, receiving surface, input material, and / or fluid (e.g., sheath fluid and / or buffer).

[0103] In some embodiments, the temperature regulation component comprises a heating element. In some embodiments, the temperature regulation component comprises a heater. In some embodiments, the temperature regulation component comprises a radiant heater, a convective heater, a conductive heater, a fan heater, a heat exchanger, or any combination thereof. In some embodiments, the temperature regulation component comprises a cooling element. In some embodiments, the temperature regulation component comprises a container of coolant, cooling liquid, ice, or any combination thereof. In some embodiments, the temperature regulation component comprises a radiant cooler, a convective cooler, a conductive cooler, a fan cooler, or any combination thereof.

[0104] In some embodiments, the temperature regulation component is configured to regulate the temperature to a set point in the range of about 0 to about 90°C, for example, about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85°C.

[0105] In some embodiments, the printing system achieves a particular geometry by moving the print head relative to a printer stage or relative to a receiving surface adapted to receive the print material. In other embodiments, the printing system achieves a particular geometry by moving the printer stage or receiving surface relative to the print head. In certain embodiments, at least a portion of the printing system is maintained in a sterile environment (e.g., within a biosafety cabinet (BSC)). In some embodiments, the printing system is configured to fit entirely within a sterile environment.

[0106] In some embodiments, the receiving surface receives excess fluid (e.g., excess sheath fluid and / or excess buffer) dispensed from the dispensing orifice and excess fluid flowing out of one or more layers of material dispensed from the dispensing orifice.

[0107] In some embodiments, the system includes components for removing excess fluid (e.g., excess sheath fluid and / or excess buffer solution) from the receiving surface onto which the fiber structure dispensed from the print head orifices is deposited, and optionally from the surface of the dispensed fiber structure. During printing, excess fluid can collect, or "pool," on the receiving surface or on the surface of the dispensed fiber structure. Such pooling can interfere with the deposition process. For example, pooled sheath fluid can cause dispensed fibers to slide off from their intended positions within the 3D structure being printed. Thus, in some embodiments, removing excess sheath fluid from the receiving surface, and optionally from the surface of the dispensed fiber structure, with a fluid removal component can improve additive manufacturing of three-dimensional structures.

[0108] Removal of excess fluid from the receiving surface or from the surface of one or more layers of dispensed fiber can be performed by drawing the fluid from these surfaces, allowing or promoting evaporation of the fluid from these surfaces, or in embodiments where the receiving surface is porous, excess fluid can be removed by drawing through the porous surface. In some embodiments, the receiving surface comprises a porous material with pores sized to facilitate fluid passage therethrough and to support one or more layers of the fibrous structure deposited thereon.

[0109] In some embodiments, a system configured to dispense material into a multiwell plate or Petri dish can include components for removing excess fluid from the receiving surface, and optionally from the surface of the dispensed fibrous structure. In some embodiments, the receiving surface on the print bed has or is positioned adjacent to an absorbent material that facilitates absorption of excess fluid from the receiving surface. For example, a well plate insert having a base made of a porous membrane material or any other porous membrane substrate can be positioned on or adjacent to an absorbent material, such as a sponge. The absorbent material acts to draw excess fluid away from the receiving surface. In embodiments in which an absorbent material is positioned below the porous receiving surface, excess fluid on the receiving surface is drawn through the porous receiving surface and into the absorbent material, thereby preventing pooling of excess fluid on the receiving surface. In embodiments in which an absorbent material is positioned immediately adjacent to or above a portion of the receiving surface (e.g., around the periphery of the receiving surface so as not to interfere with the deposition of the dispensed material), excess sheath fluid can be drawn from the receiving surface to the absorbent material.

[0110] In some embodiments, the receiving surface comprises one or more tubes fluidly coupled to a vacuum source, which can provide suction to remove excess fluid from the receiving surface and, optionally, from the surface of the dispensed fibrous structure. In such embodiments, solid or porous receiving surfaces can also be used. In some embodiments, the print head is further configured to comprise one or more vacuum channels, each having an orifice positioned near (i.e., adjacent to) the dispensing orifice. Each of the one or more vacuum channels has an inlet configured to facilitate fluid communication with one or more vacuums. When the print head is in fluid communication with the vacuum, the one or more vacuum channels apply a negative pressure to an area of ​​the receiving surface where or from which material is being dispensed from the dispensing orifice and / or to a portion of the surface area of ​​the dispensed fibrous structure, thereby wicking excess fluid from the receiving surface and, optionally, from the surface of the dispensed fibrous structure, thereby removing accumulation of fluid on the receiving surface and / or the dispensed fibrous structure.

[0111] In some embodiments, the one or more vacuum tubes are at least partially provided on one or more extensions that protrude from the print head, said extensions protruding in the same general direction as the extensions comprising the dispensing orifices and dispensing channels 110. In such embodiments, the one or more extensions comprising the vacuum tubes do not extend beyond the extensions comprising the dispensing orifices and dispensing channels 110 so as not to interfere with the dispensing process.

[0112] In some embodiments, the fluid removal feature can be a feature of the fluid composition itself. For example, the sheath fluid composition and / or buffer composition can be designed to evaporate after being dispensed from the dispensing orifice, thereby removing excess fluid buildup on the receiving surface or on the surface of the dispensed fiber structure. For example, the sheath fluid can retain a liquid state before being dispensed, but have a boiling point that leads to evaporation after being dispensed.

[0113] In some embodiments, the printing system includes a three-arm 3D motorized stage for positioning the print head and dispensing orifice in three-dimensional space above a printing bed having a surface for receiving the material to be printed. In one embodiment, the 3D motorized stage (i.e., positioning unit) can be controlled to position a vertical arm extending along the z-axis of the 3D motorized stage so that the orifice of the print head faces downward. A first horizontal arm extending along the x-axis of the motorized stage is fixed to a stationary base platform. A second horizontal arm extending along the y-axis of the motorized stage is movably coupled to the top surface of the first horizontal arm such that the longitudinal directions of the first and second horizontal arms are perpendicular to each other. It will be understood that the terms "vertical" and "horizontal" used above with respect to the arms are meant to describe how the print head moves and do not necessarily limit the physical orientation of the arms themselves.

[0114] In some embodiments, the receiving surface is positioned on top of a platform, which is coupled to the top surface of a second horizontal arm. In some embodiments, the 3D motorized stage arms are each driven by three corresponding motors and controlled by a programmable control processor, such as a computer. In a preferred embodiment, the print head and receiving surface are movable together along all three major axes of a Cartesian coordinate system by a 3D motorized stage, and the stage movement is defined using computer software. It will be understood that the present invention is not limited to the described positioning system, and other positioning systems are known in the art. As material is dispensed from the dispensing orifices on the print head, the positioning unit moves in a pattern controlled by software, thereby creating a first layer of dispensed material on the receiving surface. Additional layers of dispensed material are then stacked, so that the final 3D geometry of the dispensed material layer typically replicates the 3D geometry design provided by the software. The 3D blueprint can be created using typical 3D CAD (computer-aided design) software, as known in the art, or can be generated from a digital image. Furthermore, if the geometric forms generated by the software contain information about the specific materials used, it is possible, according to one embodiment of the present invention, to assign specific input material types to different geometric locations. For example, in some embodiments, the printed 3D structure can include two or more different input materials, each having different properties (e.g., each input material having a different cell type, different cell concentration, different ECM composition, etc.).

[0115] Aspects of the subject printing systems include software programs configured to facilitate the deposition of subject input materials in specific patterns and at specific locations to form specific fiber planar or 3D structures. To create such structures, the subject printing systems deposit the subject input materials at precise locations (two-dimensional or three-dimensional locations) on a receiving surface. In some embodiments, the locations at which the printing system deposits material are defined by user input and converted into computer code. In some embodiments, the computer code comprises a set of instructions executable by a central processing unit (CPU) of a digital processing device, written to perform a specific task. In some embodiments, printing parameters, including, but not limited to, the dimensions of the printed fiber, pump speed, travel speed of the print head positioning system, and strength or concentration of the cross-linking agent, are defined by user input and converted into computer code. In some embodiments, the printing parameters are not directly defined by user input, but are derived by the computer code from other parameters and conditions.

[0116] Aspects of the present invention include methods for producing tissue constructs, tissues, and organs, the methods including receiving, by a computer module, an input of a visual representation of a desired tissue construct; generating, by the computer module, a series of commands based on the visual representation and readable by a subject printing system; providing, by the computer module, the series of commands to the printing system; and depositing, by the printing system, one or more input materials in accordance with the commands to form a construct having a defined geometric form.

[0117] In some embodiments, the locations at which the printing system deposits input materials are defined by user input and converted into computer code. In some embodiments, the devices, systems, and methods disclosed herein further include a non-transitory computer-readable storage medium or storage medium encoded with computer-readable program code. In some embodiments, the computer-readable storage medium is a tangible component of a digital processing device, such as a bioprinter (or a component thereof) or a computer connected to the bioprinter (or a component thereof). In some embodiments, the computer-readable storage medium is optionally removable from the digital processing device. In some embodiments, computer-readable storage media include, by way of non-limiting example, CD-ROMs, DVDs, flash memory devices, solid-state memory, magnetic disk drives, magnetic tape drives, optical disk drives, and cloud computing systems and / or services. In some cases, the programs and instructions are encoded permanently, nearly permanently, semi-permanently, or non-transitoryly on the storage medium.

[0118] In some embodiments, the devices, systems, and methods described herein include software, server, and database modules. In some embodiments, a "computer module" is a software component (including code sections) that interacts with a larger computer system. In some embodiments, a software module (or program module) is provided in the form of one or more files and typically handles a specific task within a larger software system.

[0119] In some embodiments, a module is included in one or more software systems. In some embodiments, a module is integrated with one or more other modules to form one or more software systems. A computer module is optionally a standalone section of code or optionally not separately identifiable code. In some embodiments, a module resides within a single application. In other embodiments, a module resides within multiple applications. In some embodiments, a module is hosted on a single machine. In some embodiments, a module is hosted on multiple machines. In some embodiments, a module is hosted on multiple machines at a single location. In some embodiments, a module is hosted on multiple machines at multiple locations. A computer module according to embodiments of the present invention enables an end user to use a computer to perform one or more aspects of the methods described herein.

[0120] In some embodiments, the computer module comprises a graphical user interface (GUI). As used herein, "graphical user interface" refers to a user environment that uses pictorial and textual representations of application inputs and outputs and hierarchical or other data structures in which information is stored. In some embodiments, the computer module comprises a display screen. In further embodiments, the computer module presents a two-dimensional GUI via the display screen. In some embodiments, the computer module presents a three-dimensional GUI, such as a virtual reality environment, via the display screen. In some embodiments, the display screen is a touch screen and presents an interactive GUI.

[0121] Aspects of the present invention include one or more quality control components configured to monitor and / or adjust one or more parameters of the subject printing system to ensure one or more printed fibers have suitable properties. For example, in some embodiments, if the deposition process proceeds too quickly, the printed fiber structure may begin to form a coiled structure within the dispense channel 110 or outside the dispense channel 110 after being dispensed. In some embodiments, the quality control component comprises a camera configured to monitor the deposition process by collecting one or more images of the printed fiber structure and determine whether the printed fiber structure has formed a coiled structure. In some embodiments, the quality control component is configured to adjust one or more parameters of the deposition process (e.g., reduce the pressure and / or reduce the deposition rate) to reduce or avoid the formation of a coiled structure by the printed fiber structure.

[0122] Aspects of the invention include one or more fluid reservoirs configured to store fluid and deliver fluid to a printing system (e.g., a print head) through one or more fluid channels that provide fluid communication between the printing system and the reservoirs. In some embodiments, the printing system includes one or more fluid reservoirs in fluid communication with the fluid channels. In some embodiments, the fluid reservoirs are connected to input orifices of the fluid channels. In some embodiments, the fluid reservoir is configured to hold a fluid volume ranging from about 100 μL to about 1 L, e.g., about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mL, or, e.g., about 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or 950 mL.

[0123] In some embodiments, the printing system includes a pressure control unit fluidly coupled to one or more reservoirs. The pressure control unit is configured to provide a force to move one or more fluids through the printing system. In some embodiments, the pressure control unit supplies air pressure to one or more fluids through one or more connecting tubes. The applied pressure forces the fluids out of the reservoirs and through respective fluid channels to the print head. In some embodiments, alternative means may be used to move the fluids through the channels. For example, a series of electronically controlled syringe pumps may be used to provide the force to move the fluids through the print head.

[0124] In some embodiments, the printing system includes a light module (as described above) for optionally exposing the photocrosslinkable input material to crosslink the material. Light modules according to embodiments of the invention may be integrated into the print head or may be a component of the printing system.

[0125] Input materials Aspects of the present invention include input materials that can be used to print fiber structures. In some embodiments, the input material comprises a hydrogel. Non-limiting examples of hydrogels include alginate, agarose, collagen, fibrinogen, gelatin, chitosan, hyaluronic acid-based gels, or any combination thereof. Various synthetic hydrogels are known and can be used in embodiments of the systems and methods provided herein. For example, in some embodiments, one or more hydrogels form the structural basis of the printed three-dimensional structure. In some embodiments, the hydrogel has the ability to support the growth and / or proliferation of one or more types of cells, and one or more types of cells can be dispersed within the hydrogel or added to the hydrogel after it has been printed in a three-dimensional configuration. In some embodiments, the hydrogel is crosslinkable with a chemical crosslinker. For example, hydrogels containing alginate may be crosslinkable in the presence of divalent cations, hydrogels containing chitosan may be crosslinked using polyvalent anions such as sodium tripolyphosphate (STP), hydrogels containing fibrinogen may be crosslinkable in the presence of enzymes such as thrombin, and hydrogels containing collagen, gelatin, agarose, or chitosan may be crosslinkable in the presence of heat or a basic solution. In some embodiments, hydrogel fibers may be produced by a precipitation reaction, which is achieved by exposing an input material to a crosslinker material that is miscible with the input material and extracting the solvent from the input material. Non-limiting examples of input materials that form fibers via a precipitation reaction include collagen and polylactic acid. Non-limiting examples of crosslinking materials that enable precipitation-mediated hydrogel fiber formation include polyethylene glycol (PEG) and alginate. Crosslinking a hydrogel increases the hardness of the hydrogel and, in some embodiments, allows for the formation of a solidified hydrogel.

[0126] In some embodiments, the hydrogel comprises alginate. Alginate forms a solidified colloidal gel (high water content gel or hydrogel) when contacted with divalent cations. Any suitable divalent cation can be used to form a solidified hydrogel with an input material containing alginate. The alginate ion affinity series Cd 2+ >Ba 2+ >Cu 2+ >Ca 2+ >Ni 2+ >Co 2+ >Mn 2+ So, to form alginate gel, Ca 2+ has the best characteristics and is the most commonly used (Ouwerx, C. et al., Polymer Gels and Networks, 1998, 6(5):393-408). Studies have shown that Ca is mediated by poly-G blocks on adjacent polymer chains. 2+ It has been shown that calcium alginate gels, the so-called "egg box" model, form through cooperative ion bonding (ISP Alginates, Section 3: Algin - Manufacture and Structure, in Alginates: Products for Scientific Water Control, 2000, International Specialty Products: San Diego, pp. 4-7). G-rich alginates tend to form thermally stable, strong but brittle Ca gels, while M-rich alginates tend to form weaker but more elastic gels with less thermal stability. In some embodiments, the hydrogel comprises the depolymerized alginate described in U.S. Provisional Patent Application No. 62 / 437,601, the disclosure of which is incorporated herein by reference in its entirety.

[0127] In some embodiments, hydrogels can be crosslinked using free-radical polymerization reactions, which create covalent bonds between molecules. Free radicals can be generated by exposing a photoinitiator to light (often ultraviolet light) or by exposing hydrogel precursors to a chemical source of free radicals, such as ammonium persulfate (APS) or potassium peroxodisulfate (KPS) in combination with N,N,N,N-tetramethylethylenediamine (TEMED) as the initiator and catalyst, respectively. Non-limiting examples of photocrosslinkable hydrogels include methacrylated hydrogels, such as gelatin methacrylate (GEL-MA) or polyethylene (glycol) diacrylate (PEG-DA) hydrogels, which are used in cell biology because they can crosslink in the presence of free radicals after exposure to UV light and are inert to cells. PEG-DA is commonly used as a scaffold in tissue engineering because polymerization occurs rapidly at room temperature, requires little energy input, has a high water content, is elastic, and can be customized to contain various biomolecules.

[0128] Additional Components Input materials according to embodiments of the present invention can include any of a wide variety of natural or synthetic polymers that support the viability of living cells, including, for example, laminin, fibrin, hyaluronic acid, poly(ethylene)glycol-based gels, gelatin, chitosan, agarose, or combinations thereof. In particularly preferred embodiments, the subject bio-ink compositions are physiologically compatible, i.e., promote cell growth, cell differentiation, and intercellular communication. In certain embodiments, the input material includes one or more physiological matrix materials, or combinations thereof. "Physiological matrix material" refers to biological materials found in natural mammalian tissue. Non-limiting examples of such physiological matrix materials include fibronectin, thrombospondin, glycosaminoglycans (GAGs) (e.g., hyaluronic acid, chondroitin-6-sulfate, dermatan sulfate, chondroitin-4-sulfate, or keratin sulfate), deoxyribonucleic acid (DNA), adhesive glycoproteins, and collagens (e.g., collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, or collagen XVIII).

[0129] Collagen provides tensile strength to most tissues, with multiple collagen fibrils, approximately 100 nm in diameter, joining together to create strong, multi-coil fibers approximately 10 μm in diameter. The biomechanical function of a particular tissue construct is imparted through the alignment of collagen fibers in an oriented manner. In some embodiments, the input material comprises collagen fibrils. A fibrous structure can be created using an input material comprising collagen fibrils, which is then formed into a tissue construct. By adjusting the diameter of the fibrous structure, the orientation of the collagen fibrils can be controlled to direct the polymerization of the collagen fibrils in a desired manner.

[0130] For example, previous studies have shown that microfluidic channels of different diameters can direct the polymerization of collagen fibrils to form oriented fibers along the length of the channel, but only when the channel diameter is 100 μm or less (Lee et al., 2006). Primary endothelial cells grown in these oriented matrices were shown to align in the direction of the collagen fibers. In another study, Martinez et al. demonstrated that 500 μm channels in a cellulose bead scaffold can direct collagen and cell alignment (Martinez et al., 2012). In some embodiments, the input material can be formed into a fibrous structure having a diameter ranging from about 20 μm to about 500 μm, e.g., about 50 μm, about 75 μm, about 100 μm, about 125 μm, about 150 μm, about 175 μm, about 200 μm, about 225 μm, about 250 μm, about 275 μm, about 300 μm, about 325 μm, about 350 μm, about 375 μm, about 400 μm, about 425 μm, about 450 μm, or about 475 μm. By adjusting the fiber diameter, the orientation of collagen fibers within the fibrous structure can be controlled. Thus, the fibrous structure and the collagen fibers therein can be patterned to create tissue constructs with the desired configuration of collagen fibers necessary to impart desired biomechanical properties to the 3D printed structure.

[0131] Mammalian Cell Types Input materials according to embodiments of the present invention can include any mammalian cell type, including, but not limited to, stem cells (e.g., embryonic stem cells, adult stem cells, induced pluripotent stem cells), embryonic cells, endodermal cells (e.g., lung cells, liver cells, pancreatic cells, gastrointestinal cells, or urogenital tract cells), mesodermal cells (e.g., kidney cells, bone cells, muscle cells, endothelial cells, or cardiac cells), and ectodermal cells (skin cells, nervous system cells, or eye cells), or any combination thereof.

[0132] In some embodiments, the input material may include fibroblasts, chondrocytes, meniscus fibrochondrocytes, stem cells, bone marrow stromal (stem) cells, embryonic stem cells, mesenchymal stem cells, induced pluripotent stem cells, differentiated stem cells, tissue-derived cells, smooth muscle cells, skeletal muscle cells, cardiac muscle cells, epithelial cells, endothelial cells, myoblasts, chondroblasts, osteoblasts, osteoclasts, and any combination thereof.

[0133] Cells can be obtained from a donor (allogeneic) or a recipient (autologous). Cells can also be from established cell culture lines, or can be cells that have been genetically engineered and / or manipulated to obtain a desired genotype or phenotype. In some embodiments, tissue explants can also be used, which can provide several different cell types within the same structure.

[0134] In some embodiments, the cells may be obtained from a suitable human or animal donor, or from the subject into whom the cells are to be transplanted. Mammalian species include, but are not limited to, humans, monkeys, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, and rats. In one embodiment, the cells are human cells. In another embodiment, the cells may be derived from an animal such as a dog, cat, horse, monkey, or any other mammal.

[0135] Suitable growth conditions for mammalian cells are well known in the art (Freshney, RI (2000) Culture of Animal Cells, a Manual of Basic Technique. Hoboken NJ, John Wiley & Sons; Lanza et al. Principles of Tissue Engineering, Academic Press; 2nd ed. May 15, 2000; and Lanza & Atala, Methods of Tissue Engineering Academic Press; 1st ed. October 2001). Cell culture media generally contain essential nutrients and, optionally, additional elements such as growth factors, salts, minerals, vitamins, etc., which may be selected depending on the cell type(s) being cultured. Specific components may be selected to enhance cell growth, differentiation, specific protein secretion, etc. Generally, standard growth media include Dulbecco's Modified Eagle's Medium, Low Glucose (DMEM) containing 110 mg / L pyruvate and glutamine, supplemented with 10-20% fetal bovine serum (FBS) or calf serum, and 100 U / ml penicillin, as well as various other standard media known in the art. Growth conditions vary depending on the type of mammalian cell used and the tissue desired.

[0136] In some embodiments, cell-type-specific reagents may be advantageously utilized in the subject input material for use with the cell type of interest. For example, extracellular matrix ("ECM") may be extracted directly from the tissue of interest, then solubilized and incorporated into the input material to generate a tissue-specific input material for the tissue to be printed. Such ECM is readily available from patient samples and / or commercially available from suppliers such as zPredicta (rBone™, available at zpredicta.com / home / products).

[0137] activator In some aspects, input materials according to embodiments of the present invention may include at least one active agent, non-limiting examples of which include TGF-β1, TGF-β2, TGF-β3, BMP-2, BMP-4, BMP-6, BMP-12, BMP-13, basic fibroblast growth factor, fibroblast growth factor-1, fibroblast growth factor-2, platelet-derived growth factor-AA, platelet-derived growth factor-BB, platelet-rich plasma, IGF-I, IGF-II, GDF-5, GDF-6, GDF-8, GDF-10, vascular endothelial cell-derived growth factor, pleiotrophin, endothelin, nicotinamide, glucagon-like peptide-I, glucagon-like peptide-II, parathyroid hormone, tenascin-C, tropoelastin, thrombin-derived peptides, laminin, biological peptides containing cell-binding domains and biological peptides containing heparin-binding domains, therapeutic agents, and any combination thereof.

[0138] As used herein, the term "therapeutic agent" refers to any chemical moiety that is a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject. Non-limiting examples of therapeutic agents, also referred to as "drugs," are described in well-known sources such as the Merck Index, the Physician's Desk Reference, and The Pharmacological Basis of Therapeutics, and include, but are not limited to, medicines, vitamins, mineral supplements, substances used in the treatment, prevention, diagnosis, cure, or mitigation of illness or disease, substances that affect the structure or function of the body, or prodrugs that become biologically active or more active after placement in a physiological environment. In some embodiments, one or more therapeutic agents may be used that can be released from the input materials described herein into adjacent tissues or fluids upon implantation into a subject. Examples of therapeutic agents include, but are not limited to, antibiotics, anesthetics, any therapeutic agent that promotes regeneration or tissue healing, or relieves pain, infection, or inflammation, or any combination thereof.

[0139] Additional active agents may include, but are not limited to, proteins, peptides, nucleic acid analogs, nucleotides, oligonucleotides, nucleic acids (DNA, RNA, siRNA), peptide nucleic acids, aptamers, antibodies or fragments or portions thereof, antigens or epitopes, hormones, antihormones, growth factors or recombinant growth factors and fragments and variants thereof, cytokines, enzymes, antibiotics or antimicrobial compounds, anti-inflammatory agents, antifungal agents, antiviral agents, toxins, prodrugs, small molecules, drugs (e.g., drugs, dyes, amino acids, vitamins, antioxidants), or any combination thereof.

[0140] Non-limiting examples of antibiotics suitable for inclusion in the input material include aminoglycosides (e.g., neomycin), ansamycins, carbacephems, carbapenems, cephalosporins (e.g., cefazolin, cefaclor, cefditoren, ceftobiprole), glycopeptides (e.g., vancomycin), macrolides (e.g., erythromycin, azithromycin), monobactams, penicillins (e.g., amoxicillin, ampicillin, cloxacillin, dicloxacillin, flucloxacillin), polypeptides (e.g., bacitracin, polymyxin B), quinolones (e.g., ciprofloxacin, enoxacin, gatifloxacin), and the like. and ofloxacin), sulfonamides (e.g., sulfasalazine, trimethoprim, trimethoprim-sulfamethoxazole (cotrimoxazole)), tetracyclines (e.g., doxycycline, minocycline, tetracycline), chloramphenicol, lincomycin, clindamycin, ethambutol, mupirocin, metronidazole, pyrazinamide, thiamphenicol, rifampicin, thiamphenicol, dapsone, clofazimine, quinupristin, metronidazole, linezolid, isoniazid, fosfomycin, fusidic acid, or any combination thereof.

[0141] Non-limiting examples of antibodies include abciximab, adalimumab, alemtuzumab, basiliximab, bevacizumab, cetuximab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, ibritumomab tiuxetan, infliximab, muromonab-CD3, natalizumab, ofatumumab, omalizumab, palivizumab, panitumumab, ranibizumab, rituximab, tositumomab, trastuzumab, altumomab pentetate, arcitumomab, atlizumab, bectumomab, belimumab, besilesomab, biciromab, canakinumab, capromab pendetide , catumaxomab, denosumab, edrecolomab, efungumab, ertumaxomab, etaracizumab, fanolesomab, fontolizumab, gemtuzumab ozogamicin, golimumab, igovomab, imciromab, labetuzumab, mepolizumab, motavizumab, nimotuzumab, nofetumomab merpentan, oregovomab, pemtumomab, pertuzumab, rovelizumab, ruplizumab, suresomab, tacatuzumab tetraxetan, tefibazumab, tocilizumab, ustekinumab, visilizumab, votumumab, zalutumumab, zanolimumab, or any combination thereof.

[0142] Non-limiting examples of enzymes suitable for use in the input materials described herein include peroxidase, lipase, amylase, organophosphate dehydrogenase, ligase, restriction endonucleases, ribonucleases, DNA polymerases, glucose oxidase, and laccase.

[0143] Additional non-limiting examples of active agents suitable for use with the subject input materials include cell growth media such as Dulbecco's modified Eagle's medium, fetal bovine serum, non-essential amino acids, and antibiotics; growth factors and morphogenetic factors such as fibroblast growth factor, transforming growth factor, vascular endothelial growth factor, epidermal growth factor, platelet-derived growth factor, insulin-like growth factor, bone morphogenetic growth factor, bone morphogenetic-like protein, transforming growth factor, nerve growth factor, and related proteins (growth factors are known in the art, see, e.g., Rosen & Thies, CELLULAR & MOLECULAR BASIS BONE FORMATION & REPAIR, RG Landes, 1999); Co., Austin, Tex., 1995), anti-angiogenic proteins such as endostatin and other naturally occurring or genetically engineered proteins, polysaccharides, glycoproteins, or lipoproteins, anti-infective agents such as antibiotics and antivirals, chemotherapeutic agents (i.e., anti-cancer agents), anti-rejection agents, analgesics and analgesic combinations, anti-inflammatory agents, steroids, or any combination thereof.

[0144] Additional Fluids

[0145] Aspects of the invention include one or more buffers. Buffers according to embodiments of the invention are miscible with the input material (e.g., hydrogel) and do not crosslink the input material. In some embodiments, the buffer comprises an aqueous solvent. Non-limiting examples of buffers include polyvinyl alcohol, water, glycerol, propylene glycol, sucrose, gelatin, or any combination thereof.

[0146] Buffers according to embodiments of the invention can have a viscosity ranging from about 1 mPa·s to about 5,000 mPa·s, e.g., about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,250, 3,500, 3,750, 4,000, 4,250, 4,500, or 4,750 mPa·s. In some embodiments, the viscosity of the buffer can be adjusted to match the viscosity of one or more input materials.

[0147] Aspects of the present invention include one or more sheath fluids. A sheath fluid according to embodiments of the present invention is a fluid that can be used to at least partially surround or "sheath" an input material dispensed from dispensing channel 110. In some embodiments, the sheath fluid comprises an aqueous solvent. Non-limiting examples of sheath fluids include polyvinyl alcohol, water, glycerol, propylene glycol, sucrose, gelatin, or any combination thereof. Sheath fluids according to embodiments of the invention can have viscosities ranging from about 1 mPa·s to about 5,000 mPa·s, such as about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,250, 3,500, 3,750, 4,000, 4,250, 4,500, or 4,750 mPa·s. In some embodiments, the viscosity of the sheath fluid can be adjusted to match the viscosity of one or more input materials.

[0148] In some embodiments, the sheath fluid comprises a chemical crosslinker. In some embodiments, the chemical crosslinker comprises a divalent cation. Non-limiting examples of divalent cations include Cd 2+ , Ba 2+ , Cu 2+ , Ca 2+ , Ni 2+ , Co 2+ , or Mn2+ In a preferred embodiment, Ca 2+ is used as the divalent cation. In some embodiments, the concentration of the divalent cation in the sheath fluid is in the range of about 80 mM to about 140 mM, for example, about 90, 100, 110, 120, or 130 mM.

[0149] How to use Aspects of the present invention include methods for printing linear fiber structures, planar structures comprising one or more fiber structures, or three-dimensional (3D) structures comprising two or more layers of planar structures. In some embodiments, the method first includes providing a design of the planar or 3D structure to be printed. The design can be created using commercially available CAD software. In some embodiments, the design includes information regarding specific materials to assign to specific locations within the structure(s) to be printed (e.g., in the case of heterogeneous structures comprising multiple materials).

[0150] In some embodiments, the method includes using a 3D printer, the printer including a print head, a receiving surface that receives material dispensed by the print head, and a positioning unit operatively connected to the receiving surface that positions the print head at a location in three-dimensional space above the receiving surface. For example, various embodiments of the printing system provided herein may be used in methods for printing flat surfaces or 3D structures.

[0151] Aspects of the method include providing one or more input materials that the print head dispenses. In some embodiments, one or more cell types are compatible with and optionally formulated within the input materials. In some embodiments, a sheath fluid functions as a lubricant to lubricate the movement of the input materials within the print head. In some embodiments, the sheath fluid includes a crosslinker that solidifies at least a portion of the hydrogel before or while the hydrogel is dispensed from the print head.

[0152] Aspects of the method include communicating the design to a 3D printer. In some embodiments, the communication may be accomplished, for example, by a programmable control processor. In some embodiments, the method includes controlling the relative positioning of the print head and the receiving surface in three-dimensional space while simultaneously dispensing a sheath fluid and a dosing material, alone or in combination, from the print head. In some embodiments, the materials dispensed from the print head are dispensed coaxially, such that the sheath fluid envelops the dosing material. This coaxial configuration allows a cross-linking agent in the sheath fluid to solidify the dosing material, thereby generating a solidified fiber structure that is dispensed from the print head.

[0153] In some embodiments, the method includes depositing a first layer of a dispensed fiber structure onto a receiving surface, the first layer including a fiber structure configuration specified by a blueprint, and repeating the depositing step to deposit subsequent fiber structures onto the first layer and subsequent layers, thereby depositing layers upon layers of the dispensed fiber structure in the geometric configuration specified by the blueprint to create a 3D structure.

[0154] In some embodiments, multiple input materials, e.g., multiple hydrogels, at least some of which contain one or more cell types, are deposited in a controlled sequence, allowing for controlled placement of the deposited input materials and cell types in geometric configurations dictated by a blueprint.

[0155] In some embodiments, the method includes removing excess fluid from the receiving surface and, optionally, from the surface of the dispensed fibrous structure. For example, the step of removing excess fluid can be performed continuously throughout the printing process, thereby removing excess fluid that would otherwise interfere with the deposition of the dispensed fibrous structure in the geometric configuration provided by the design. Alternatively, the step of removing excess fluid can be performed intermittently throughout the printing process, either continuously with or simultaneously with one or more deposition steps. In some embodiments, the removal of excess fluid is achieved by withdrawing fluid from the receiving surface and, optionally, from the surface of the dispensed fibrous structure. In some embodiments, the removal of excess fluid is achieved by withdrawing excess fluid through a receiving surface having pores sized to allow the passage of fluid. In some embodiments, the removal of excess fluid is achieved by providing a fluid that evaporates after being dispensed from a dispensing orifice.

[0156] Embodiments of the present invention include methods for fabricating 3D structures comprising one or more input materials, which are utilized to repair and / or replace at least a portion of damaged or diseased tissue in a subject.

[0157] As mentioned above, Cd 2+ , Ba 2+ , Cu 2+ , Ca 2+ , Ni 2+ , Co 2+ , or Mn 2+ Any suitable divalent cation may be used in conjunction with the subject method to solidify the chemically crosslinkable input material, including, but not limited to, Ca. In a preferred embodiment, Ca 2+ is used as the divalent cation. In one preferred embodiment, the chemically crosslinkable input material is Ca 2+ In some embodiments, the sheath fluid is contacted with a solution containing Ca to form a solidified fiber structure. 2+ The concentration of is in the range of about 80 mM to about 140 mM, for example, about 90, 100, 110, 120, or 130 mM.

[0158] In certain embodiments, the dosage material solidifies in less than about 5 seconds, such as less than about 4 seconds, less than about 3 seconds, less than about 2 seconds, or less than about 1 second.

[0159] Aspects of the invention include methods for depositing one or more input materials in a patterned manner using software tools to form layers of solidified structures that are formed into multi-layered 3D tissue structures. In some embodiments, the multi-layered 3D tissue structures comprise a plurality of mammalian cells. Advantageously, by adjusting the components of the subject input materials (e.g., mammalian cell type, cell density, matrix components, active agents), the subject methods can be used to create multi-layered 3D tissue structures that have precisely controlled compositions at any particular location in three-dimensional space. Thus, the subject methods facilitate the generation of complex three-dimensional tissue structures.

[0160] In some embodiments, the method includes simultaneously dispensing a buffer and / or sheath fluid through the core channel, one or more input materials through one or more shell channels, and a sheath fluid through the sheath flow channel 118 to form a hollow core in the printed fiber.

[0161] In some embodiments, the non-crosslinkable material in the core channel comprises a buffer solution, and the sheath fluid in the sheath flow channel 118 comprises a chemical crosslinker, and contact occurs at the sheath fluid intersection, solidifying the outer surface of the flow of crosslinkable material in the dispensing channel 110.

[0162] In some embodiments, the non-crosslinkable material in the core channel comprises a chemical crosslinker, the sheath fluid in the sheath flow channel 118 comprises an aqueous solvent, and contact occurs at the first fluid focusing intersection, causing the inner surface of the flow of crosslinkable material in the dispensing channel 110 to solidify.

[0163] In some embodiments, the non-crosslinkable material in the core channel comprises a chemical crosslinker, the sheath fluid in the sheath flow channel 118 comprises a chemical crosslinker, contact occurs at the first fluid focusing intersection and the inner surface of the flow of crosslinkable material solidifies in the dispensing channel, and contact occurs at the sheath fluid intersection and the outer surface of the flow of crosslinkable material solidifies in the dispensing channel 110.

[0164] In some embodiments, the system includes a print head including a core channel including at least two core inlet subchannels connected to separate fluid reservoirs containing different input materials, and the method includes alternately dispensing the different input materials through the shell inlet subchannels 126 to produce a solidified fiber structure containing different core materials along the length of the continuous fiber.

[0165] In some embodiments, the system includes a print head including a first shell channel including at least two shell inlet subchannels 126 connected to separate fluid reservoirs containing different materials, and the method includes alternately dispensing different input materials through the shell inlet subchannels 126 to produce a solidified fiber structure including different shell materials along the length of the continuous fiber.

[0166] In some embodiments, the system includes a print head including at least two shell channels connected to separate fluid reservoirs containing different input materials, and the method includes simultaneously dispensing the different input materials through the first and second shell channels to create a solidified fiber structure including different concentric shells.

[0167] In some embodiments, the system includes a print head including a first shell channel including at least two shell inlet sub-channels 126 connected to separate fluid reservoirs containing a reinforced hydrogel material and a biocompatible hydrogel material, and the method includes alternately dispensing the reinforced hydrogel material and the biocompatible hydrogel material via the dispensing channel 110 to generate a perfusable tissue fiber. In another embodiment, the print head further includes a second shell channel 128, and the method includes dispensing the same or a different reinforced hydrogel material via the second shell channel 128 to generate a second shell concentric about the first shell.

[0168] In an alternative embodiment, solid-core fibers can be created as a means of printing materials that would otherwise be unprintable. This method allows for the selection of an easily printable shell material, such as alginate, and an otherwise unprintable core material, such as pure collagen. Here, the ability to switch core materials allows the user further control over the core composition. The core material may contain different cell types, arranged along the length of the fiber, or combined. In this case, the shell material can also be switched and contain different cell types.

[0169] Practicality In some embodiments, structures generated using the systems and methods provided herein may be useful, for example, in the field of drug discovery, where identifying cellular responses to various compounds and compositions is important. The use of planar and 3D cell cultures generated using embodiments of the systems and methods provided herein can provide experimental conditions that more closely resemble in vivo cell and tissue conditions compared to traditional 2D cell cultures. The 3D organization of cells can more closely mimic in vivo cell-to-cell interactions and responses to external stimuli, and the hybrid nature of 3D structures that can be generated using the systems and methods provided herein enables the study of tissues and potentially organs. It is believed that 3D cell-containing structures generated using embodiments of the systems and methods provided herein could provide similar benefits to the cosmetics industry by providing an alternative means for testing cosmetics.

[0170] In some embodiments, various aspects of the systems and methods provided herein are compatible with standard well-plate technology. Well plates or well-plate inserts may be used in the methods and systems provided herein in conjunction with or as part of the print bed. Thus, various embodiments of the systems and methods provided herein are compatible with equipment and practices that utilize well plates, allowing them to be easily integrated into existing process flows.

[0171] In some embodiments, one or more fluid channels within the subject print heads are compatible with other microfluidic modules. For example, known microfluidic modules can be included in the print heads of the systems provided herein upstream of the dispensing orifices. Such modules can include, for example, a cell counting module, a cell sorting module, a cell analysis module, and / or a concentration gradient generation module.

[0172] In some embodiments, 3D printing throughput can be increased by adding additional print heads to the system in parallel. Because each print head contains all of the elements necessary to print a multi-material structure, including additional print heads in the system allows for several 3D structures to be printed simultaneously.

[0173] All patents and patent publications mentioned herein are incorporated by reference in their entirety.

[0174] Although the foregoing invention has been described in some detail by way of illustration and example for clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the present invention that certain changes and modifications thereto can be made without departing from the spirit or scope of the appended claims.

[0175] Because the subject invention allows for multiple material switching, the composition of the vessel wall (cell type and biomaterial composition) can be varied along the length of the channel while printing continuously. An example of this is recapitulating the biological structure and function of a renal tubule, where the wall composition at the proximal end is different from that at the distal end. Or, in the case of printed perfusable 3D liver tissue, the vessel wall at the larger, open end of the vessel may be lined with low-permeability portal arteriolar endothelial cells, while further inside the vessel, where the conduit narrows to form sinusoids, the vessel wall may be lined with more permeable sinusoidal endothelial cells. Similar to liver tissue, it is desirable to investigate the interaction between the contents of the perfused channel and different types of interstitial cells outside the channel in one or more of the outer shells. This can be applied to generate multi-tissue models of toxicity combined with the effects of shear flow. A single tissue can be printed by switching the cellular content of the fiber's shell along its length to generate coded hollow fibers with different regions corresponding to different organ types. This switching of shell contents is not possible with non-microfluidic syringe-based systems. [Example]

[0176] Example 1: Perfusable tissue fibers Bioprinted perfusable tissue fibers with a liquid core and a cell-containing gel shell that can be printed and then attached to a pump that inserts needles into the fiber core and pushes the fluid of interest through the fiber are of great commercial and clinical interest. In this way, the flow of nutrients, drugs, or other compounds of interest through the cell-containing fiber can be simulated. Unfortunately, connecting needles to the fiber presents significant challenges, as the mechanical requirements for making such connections are significantly different from those necessary to support functional biological functions. Therefore, switching the shell material in real time during printing allows users to print stronger materials in the areas intended for needle connection (the fiber ends) and softer, cell-containing materials in the areas intended to support biological functions.

[0177] As shown in Figure 12, perfusable tissue fibers according to the subject invention were bioprinted with a reinforced hydrogel material (blue) consisting of 4 wt% low-viscosity sodium alginate, a biocompatible hydrogel material (red) consisting of 1.3 wt% of the same alginate, and a liquid core composed of 3% polyvinyl alcohol. A standard 30-gauge stainless steel Luer-lock needle was then used to connect the fiber, and a mixture of gelatin and transglutaminase was used to seal the needle / fiber connection and prevent leakage during perfusion.

[0178] Furthermore, even with adequate reinforcement at the ends of perfusable fibers to allow proper connection, rupture may still occur along the length of fibers containing softer cell-containing materials, especially at higher flow rates. Thus, as shown in Figure 13, the present invention further contemplates the optional addition of a second concentric shell layer containing the same or a different reinforcing hydrogel material so that the fiber is supported along its entire length.

[0179] The foregoing merely illustrates the principles of the present invention. It will be appreciated by those skilled in the art that, although not explicitly described or shown herein, they will be able to devise various configurations which embody the principles of the present invention and are within its spirit and scope. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader's understanding of the principles of the present invention and the concepts contributed by the inventors to further this technology, and should not be construed as being limited to such specifically recited examples and conditions. Furthermore, all statements herein reciting principles, aspects, and specific examples thereof are intended to encompass both structural and functional equivalents. Furthermore, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed to perform the same function, regardless of structure. Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied by the appended claims.

Claims

1. A print head for a 3D printer, The print head includes a plurality of stacked layers that form a plurality of fluid channels, and the plurality of fluid channels are A core channel having at least one inlet and one outlet, A first shell channel having at least one inlet and one outlet, A first fluid focusing chamber that converges toward the dispensing channel, Includes, The core channel and / or the first shell channel further comprises at least one fluid distribution orifice configured to distribute fluid around the outlet of the core channel and / or the outlet of the first shell channel, The at least one fluid distribution orifice connects the core channel inlet and / or the first shell channel inlet to the vertex of the upper curved surface of the core channel outlet and / or the first shell channel outlet, and the upper curved surface has a parabolic or elliptical shape. The core channel outlet is in fluid communication with the inlet of the first fluid focusing chamber and is aligned with the dispensing channel. The first shell channel outlet is arranged concentrically around the core channel and is in fluid communication with the inlet of the first fluid focusing chamber. The first fluid focusing chamber is configured to converge toward the dispensing channel and to focus the fluid toward the dispensing channel. Printhead.

2. Furthermore, the second shell channel has at least one inlet, one outlet, and at least one fluid distribution orifice configured to distribute fluid around the outlet of the second shell channel, The print head according to claim 1, wherein the second shell channel outlet is concentrically arranged around the first shell channel outlet within the same layer of the print head and is in fluid communication with the first fluid focusing chamber.

3. Furthermore, the second shell channel has at least one inlet, one outlet, and at least one fluid distribution orifice configured to distribute fluid around the outlet of the second shell channel, A second fluid focusing chamber is located between the first fluid focusing chamber and the distal end of the dispensing channel, Equipped with, The second shell channel outlet is arranged concentrically around the dispensing channel and is in fluid communication with the inlet of the second fluid focusing chamber. The print head according to claim 1, wherein the second fluid focusing chamber is configured to converge toward the dispensing channel and to focus the fluid toward the dispensing channel.

4. The print head according to claim 1, wherein the first fluid focusing chamber and / or the second fluid focusing chamber have a frustoconical shape.

5. The print head according to claim 2 or 3, wherein the at least one fluid distribution orifice connects the inlet of the second shell channel to the vertex of the upper curved surface of the outlet of the second shell channel, and the upper curved surface has a parabolic or elliptical shape.

6. The printhead according to claim 2 or 3, wherein the first and / or second shell channel inlet includes two or more subchannels configured to supply fluid to the same or separate fluid distribution orifices.

7. Each subchannel includes a fluid distribution orifice connecting the inlet of the first and / or second shell channel to the vertex of the upper curved surface of the respective first and / or second shell channel outlet. The print head according to claim 6, wherein the upper curved surface has a parabolic or elliptical shape.

8. The print head according to claim 6 or 7, wherein each subchannel is configured to dispense a different material.

9. The print head according to claim 1, further, A sheath fluid chamber located between the first fluid focusing chamber and the distal end of the dispensing channel is provided with a sheath flow channel that converges with the dispensing channel. The sheath fluid chamber converges toward the dispensing channel. Printhead.

10. Furthermore, the sheath fluid chamber located between the first and second fluid focusing chambers and the distal end of the dispensing channel is provided with a sheath flow channel that converges with the dispensing channel. The print head according to claim 2 or 3, wherein the sheath fluid chamber converges toward the dispensing channel.

11. The print head according to claim 9 or 10, wherein the sheath fluid chamber has a frustoconical shape configured to focus the fluid toward the dispensing channel.

12. The printhead according to claim 9 or 10, wherein the sheath flow channel includes a plurality of sheath flow subchannels that converge toward the dispensing channel through the sheath fluid chamber.

13. The print head according to claim 1, wherein the print head comprises at least two core inlet subchannels that converge at or near the core channel outlet and / or the fluid distribution orifice.

14. The printhead according to claim 13, wherein the at least two core inlet subchannels converge in the same printhead layer as the core channel outlet and / or the fluid distribution orifice, or in the layer immediately preceding it.

15. The printhead according to claim 13 or 14, wherein each core inlet subchannel is configured to dispense a different material.

16. A system for generating fiber structures, A print head according to any one of claims 1 to 15, A system comprising a positioning component for positioning the dispensing orifice of the print head in three-dimensional space, wherein the positioning component is operably coupled to the print head.

17. The system according to claim 16, further comprising a programmable control processor for controlling the positioning components and controlling the flow rate of one or more fluids passing through the print head.

18. Furthermore, it includes a fluid removal component configured to remove excess fluid dispensed from the print head, The fluid removal component includes a porous membrane configured to allow the excess fluid to pass through, and / or includes a vacuum configured to suck up the excess fluid. The system according to claim 16 or 17.

19. The system according to any one of claims 16 to 18, further comprising a pressure control component configured to adjust the flow rate of one or more fluids passing through the print head.

20. A method for generating a core-shell fiber structure, The aforementioned method, The process includes providing a system for generating a fibrous structure, the system being A print head according to any one of claims 1 to 15, wherein the print head is configured to dispense a plurality of input materials through the core channel and the shell channel, wherein at least one of the input materials comprises a crosslinkable material, and the print head is configured to dispense a sheath solution through the sheath flow channel, A receiving surface for receiving the first layer of material dispensed from the print head, A positioning component for positioning the dispensing orifice of the print head in three-dimensional space, comprising a positioning component operably coupled to the print head, A programmable control processor for controlling the positioning components and controlling the flow rate of one or more fluids passing through the print head, A fluid reservoir containing the plurality of input materials and sheath solution, the fluid reservoir being in fluid communication with the print head, Equipped with, A step of generating a solidified fibrous structure by bringing the crosslinkable material into contact with the sheath solution within the dispensing channel, The process of dispensing the solidified fibrous structure from the dispensing orifice of the print head, Methods that include...

21. The system comprises a core channel including at least two core inlet subchannels connected to separate fluid reservoirs, each containing a first and a second input material, The method according to claim 20, further comprising the step of generating a solidified fiber structure containing different core materials along the longitudinal direction of a continuous fiber by alternately dispensing the first and second input materials through the at least two core inlet subchannels.

22. The system comprises a first shell channel including at least two shell inlet subchannels connected to separate fluid reservoirs, each containing a third and a fourth input material, The method according to claim 20 or 21, further comprising the step of generating a solidified fiber structure containing different shell materials along the longitudinal direction of a continuous fiber by alternately dispensing the third and fourth input materials through the shell inlet subchannel.

23. The system comprises first and second shell channels connected to separate fluid reservoirs containing third and fourth input materials, respectively. The method according to claim 20 or 21, further comprising the step of generating a solidified fibrous structure containing different concentric shells by dispensing the third and fourth input materials through the first and second shell channels.

24. Furthermore, the process includes encoding the planar structure to be printed into the programmable control processor, The method according to claim 20, comprising the step of depositing the first layer of the solidified fibrous structure onto the receiving surface to print the planar structure.

25. Furthermore, the process includes encoding the 3D structure to be printed into the programmable control processor, The method according to claim 24, comprising the step of depositing a subsequent layer of the solidified fiber structure on the planar structure to print a 3D structure.

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