Systems and methods for printing core-shell fibers

Abstract

A printhead for a three-dimensional printer, in one embodiment, comprises a multi-channel housing comprising a core channel outlet, a first shell channel outlet, and a first fluid focusing chamber converging toward a distribution channel, wherein the core channel outlet is in a central region of the housing, and the core and shell channels extend into the housing to respective depths. In another embodiment, the plurality of shell channels comprises an inner shell channel that extends longer into the focusing chamber than the outer shell channels, and the core channel extends longer into the focusing chamber than any of the shell channels. In another embodiment, each of the core channel and the first shell channel comprises at least two inlet subchannels having distinct fluid reservoirs, input orifices, and control valves that converge to form a single outlet in fluid communication with the respective focusing chamber. A sheath flow channel may be provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to the filing dates of U.S. Provisional Patent Application Serial No. 62 / 929,720, filed on November 1, 2019, and U.S. Provisional Patent Application Serial No. 63 / 030,885, filed on May 27, 2020, the disclosures of which are incorporated herein by reference in their entireties. Technical Field

[0003] The present invention relates to systems and methods for producing core-shell fiber structures and to three-dimensional (3D) printing of such structures from digital files. In some embodiments, the printed fibers include living cells. Background of the Invention

[0005] Tissue engineering has long sought to create viable synthetic structures capable of mimicking and / or replacing living organs and tissues using a myriad of materials and methods. The lack of pre-patterned vasculature is one of the major factors limiting the success of current tissue engineering strategies, and the current inability to fabricate thick tissue constructs containing endogenous engineered vasculature or nutrient pathways that can integrate with host tissue is a major technical barrier preventing the generation and / or implantation of larger, viable, and / or metabolically active tissues.

[0006] 3D printing is a form of additive manufacturing that 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. Initial efforts to adapt these 3D printing technologies for creating hollow vessel patterned structures focused on printing and subsequently eliminating sacrificial materials. Bertassoni et al., for example, used a physical method to remove templated agarose from a surrounding cast of a photocrosslinked acrylic hydrogel (e.g., gelMA). (Lab on a Chip 14:2202 (2014)). The printed agarose fibers had minimal adhesion to the gelMA but unfortunately had to be removed manually, which was time-consuming and difficult and also required the cast hydrogel to be stronger than the agarose fibers.

[0007] Alternative approaches involve printing a sacrificial fiber network from a material that can subsequently be removed by solubilization or liquefaction. For example, Wu et al. printed a 3D perfusable vascular tree by extruding sacrificial Pluronic F127 filaments within a Pluronic F127-diacrylate gel reservoir to provide support during printing (Advanced Materials 2011;23:H178-183). After photocuring of the surrounding acrylate-modified Pluronic F127-diacrylate, the unmodified Pluronic F127 channels could be liquefied by lowering the temperature below their critical micelle temperature, leaving perfusable channels. Using a similar approach, Lee et al. deposited a collagen support matrix layer around gelatin containing human umbilical vein endothelial cells (HUVECs) (Biomaterials 2014;35:8092–8102). After printing, the gelatin melted, which facilitated cell seeding of the “activated” HUVECs onto the surrounding collagen. Various other sacrificial materials have also been printed, including the use of "carbohydrate glass" as a sacrificial material by Miller et al., which showed subsequent infusion of a hollow network (Bio-Med 2012; 11: 768-774).

[0008] However, to date, Pluronic 127’s liquefaction properties at low temperatures have made it the most commonly used sacrificial material, and Kolesky and colleagues have successfully used it with various support materials to create thick, vascularized tissue constructs. (Advanced Materials 2014;26:3124–3130) (Co-printed channel structures of Pluronic F127 and cell-laden gelatin methacrylate (GelMA); Proceedings of the National Academy of Sciences of the United States of America 2016;113:3179–3184) (Pluronic F127 mixed with thrombin, designated as a “vascular ink,” was used to indirectly print sacrificial channels within cell-laden gelatin fibrinogen bioink). However, it is important to note that sacrificial materials such as Pluronic F127 are cytotoxic at higher concentrations, and it is unclear what effect liquefied Pluronic will have on the surrounding tissue, as it is unlikely to be completely removed from the hollow channels.

[0009] A newer alternative to sacrificial hollow fiber patterning is to use a focused laser beam to heat and ablate areas within a precast (or printed) tissue structure. As the laser beam moves, it leaves behind a hollow tunnel. The technique can be relatively fast and can 3D pattern branched hollow tubes with high resolution, potentially with capillaries as small as 10-20 microns in diameter. A 2-photon laser can be used to increase the penetration depth of the beam, which also helps reduce the intensity of out-of-focus light, thereby reducing phototoxicity to areas outside the ablation channel.

[0010] Direct bioprinting of hollow tubes within larger tissues has also been attempted. For example, Gao et al. demonstrated the ability to generate and print hollow alginate fibers using a coaxial needle with a calcium chloride crosslinking solution within the alginate fiber core. The printing nozzle was configured for internal flow of the calcium solution and external flow of the alginate solution (i.e., bioink), resulting in a construct with endogenously perfusable microchannels. In this approach, hollow microchannels were printed in stages that were gradually lowered into a calcium bath solution for secondary crosslinking. (Biomaterials 2015;61:203–215). Hinton et al. developed an alternative to liquid immersion printing that employed an extrusion method using various hydrogels to support direct structure printing in a sacrificial gelatin-microparticle bath to facilitate crosslinking. (Science Advances 2015;1:e1500758).

[0011] Unfortunately, however, the above-mentioned systems, devices and materials for conventional 3D bioprinting of hollow fiber networks have many shortcomings that hinder their more practical, effective and widespread implementation. As mentioned above, manual (physical) removal of sacrificial materials is impractical, inconsistent, time-consuming, and may be impossible for smaller containers. In addition, patterning vascular channels with sacrificial materials limits the ability to pattern cells and / or biomaterials in an axial manner around the hollow channels. It is difficult to imagine how this technology can be used to manufacture, for example, patterned channel networks that simulate real arteriolar structures with smooth muscle cells around an endothelial cell inner layer.

[0012] With laser ablation, penetration depth is limited to 1mm to 2mm, and optically transparent materials that do not scatter the beam are required, whereas most cellular tissues are opaque and light-scattering. Finally, with extrusion printing of sacrificial materials, the diameter of the sacrificial fiber (and subsequently the inner diameter of the channel) is determined by the diameter of the extrusion needle. This diameter is fixed, so there is no opportunity to dynamically change the lumen diameter of the channel in different areas of the tissue.

[0013] Therefore, there is a need for systems and devices that can dispense and pattern hollow channels within 3D tissues, where pro-angiogenic bioinks and different cell types are precisely arranged axially and parallel to the channels. The technology should be compatible with cell viability, and the inner diameter of the printed channels should be dynamically modified from capillaries to larger vessels within a single tissue structure. For example, it may be desirable to have larger diameter vessels at the tissue opening where a perfusion device is attached, and then reduce the lumen diameter to mimic smaller vessels within the tissue. Modifying vessel diameter may also be a useful tool to modulate blood flow changes and restrictions in diseases such as atherosclerosis. The present invention addresses these and other unmet needs. Summary of the Invention

[0014] Aspects of the present invention include systems and methods for producing core-shell fiber structures including hollow fibers and multi-shell structures and for producing three-dimensional (3D) structures from digital files. In some embodiments, the printed fibers include living cells. As demonstrated herein, direct printing of core-shell fibers using the present invention can produce fibers with different diameters and multiple shells, and different cell types can be loaded into different shells in precise axial and parallel arrangements to produce hollow blood vessels with multiple cell layers. In addition, the composition of the vessel wall (cell type and biomaterial composition) can be modified along the length of the channel while printing continuously.

[0015] Aspects of the present invention include a microfluidic printhead for producing a core-shell fiber structure, the printhead comprising a plurality of stacked and preferably bonded layers forming a plurality of flow paths, the printhead comprising: at least one core channel having at least one inlet 100 and an outlet 102 and one or more fluid switches; a first shell channel having at least one inlet and an outlet; at least one multi-channel housing 108; and a distribution channel 110, wherein the multi-channel housing 108 comprises the core channel outlet 102, the first shell channel outlet 106, and a first fluid focusing chamber 112; wherein the core channel outlet 102 is disposed in the multi-channel housing 108. 108, wherein the core channel outlet 102 extends to a first vertical depth in the multichannel housing 108, preferably wherein the core channel outlet 102 extends to a first vertical depth in the first fluid focusing chamber 112, aligned with the distribution channel 110; wherein the first shell channel outlet 106 is concentrically disposed about the core channel, is in fluid communication with the inlet of the first fluid focusing chamber 112, and extends to a second vertical depth in the multichannel housing 108; and wherein the first fluid focusing chamber 112 converges toward the distribution channel 110, preferably wherein the first fluid focusing chamber 112 comprises a frustoconical shape configured to focus fluid toward the distribution channel 110. In some embodiments, the first shell channel outlet 106 has a gradient width that increases with increasing depth into the multichannel housing 108. In an exemplary embodiment, the first shell channel outlet 106 comprises a hollow cylinder having an axis of rotation that does not intersect the core channel outlet 102.

[0016] In some embodiments, wick channel outlet 102 extends through a majority of the length of multi-channel housing 108. In a preferred embodiment, the first vertical depth is greater than the second vertical depth, such that wick channel outlet 102 extends further into multi-channel housing 108 and / or first fluid focusing chamber 112 than first shell channel outlet 106. In an alternative embodiment, the second vertical depth is greater than the first vertical depth, such that first shell channel outlet 106 extends further into multi-channel housing 108 than wick channel outlet 102. In some embodiments, first fluid focusing chamber 112 is located on a separate layer of the printhead, and first shell channel outlet 106 extends from a previous layer of the printhead into a first fluid focusing chamber 112 in an adjacent downstream layer of the printhead.

[0017] In some embodiments, the printhead includes at least two wick channels that converge to form a wick channel outlet 102 in fluid communication with a first fluid focusing chamber 112. In a preferred embodiment, the at least two wick channels converge at or near the wick channel outlet 102, the multichannel housing 108, and / or a fluid dispensing orifice as further described herein. In a particularly preferred embodiment, the at least two subchannels converge in the immediately preceding layer, or in the same layer of the printhead as the wick channel outlet 102, the multichannel housing, and / or the fluid dispensing orifice. In one embodiment, the wick channel is configured to dispense a non-crosslinkable material. In an exemplary embodiment, the first wick channel includes a sheath fluid input orifice and a control valve, and the second wick channel includes a buffer solution input orifice and a control valve.

[0018] In some embodiments, the printhead further comprises a second shell channel 128 having at least one inlet and an outlet, wherein the second shell channel outlet and / or inlet 132, 106 are concentrically disposed about the first shell channel outlet 106 in the multi-channel housing 108 in the same layer of the printhead and in fluid communication with the first fluid focusing chamber 112. In a preferred embodiment, the first shell channel outlet 106 extends further into the multi-channel housing 108 than the second shell channel outlet 132. In some embodiments, the first fluid focusing chamber 112 is located on a separate layer of the printhead, and the first shell channel outlet 106 and / or the second shell channel outlet 132 extend from a previous layer of the printhead into the first fluid focusing chamber 112 in an adjacent downstream layer of the printhead.

[0019] In an alternative embodiment, the printhead further comprises: a second shell channel 128 having at least one inlet and an outlet; and a second multi-channel housing 134 positioned between the first fluid focusing chamber 112 and a distal end of the distribution channel 110, wherein the second multi-channel housing 134 comprises the distribution channel 110, the second shell channel outlet 132, and the second fluid focusing chamber 136; wherein the distribution channel 110 is disposed in a central region of the second multi-channel housing 134, is in fluid communication with the inlet of the second fluid focusing chamber 136, and extends to a first vertical depth into the multi-channel housing, preferably wherein the second shell channel outlet 132 is concentrically disposed about the distribution channel 110, is in fluid communication with the inlet of the second fluid focusing chamber 136, and extends to a second vertical depth into the multi-channel housing; and wherein the second fluid focusing chamber 136 converges toward the distribution channel 110, preferably wherein the second fluid focusing chamber 136 comprises a frusto-conical shape configured to focus fluid toward the distribution channel 110. In some embodiments, the first multi-channel housing 108 and the second multi-channel housing 134 are located in consecutive layers of the printhead. In other embodiments, the first and second multi-channel housings, or portions thereof, can be located in the same layer of the printhead; for example, the second multi-channel housing can overlap with a first fluid focusing chamber in the same layer of the printhead. In some embodiments, the second fluid focusing chamber 136 is located in a separate layer of the printhead, and the second housing channel outlet 132 extends from a previous layer of the printhead into the second fluid focusing chamber 136 in an adjacent downstream layer.

[0020] 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 circumference of the first shell channel outlet 106 and / or the second shell channel outlet 132. In one embodiment, the fluid distribution orifice connects the first shell channel inlet 104 and / or the second shell channel inlet 130 with the vertex 116 of the upper curved surface 114 of the first shell channel outlet 106 and / or the second shell channel outlet 132, preferably wherein the upper curved surface 114 of the first shell channel outlet 106 and / or the second shell channel outlet 132 has a parabolic or elliptical shape. In an exemplary embodiment, the first shell channel outlet 106 and / or the second shell channel outlet 132 comprises a truncated hollow cylinder having an elliptical upper surface, with the vertex 116 positioned at the fluid distribution orifice.

[0021] In some embodiments, the first shell channel inlet 104 and / or the second shell channel inlet 130 are configured to dispense two different materials, including two different hydrogel materials and / or two different porous materials, so that the composition of the first and / or second shell layer can change along the length of the printed fiber. In some embodiments, the first shell channel inlet 104 and / or the second shell channel inlet 130 include different subchannels with different 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 channel may include two fluid switches, and each subchannel may be fluidically connected to a different fluid switch. In another embodiment, the core channel may include two fluid switches, and each core inlet subchannel may be fluidically connected to a different fluid switch.

[0022] In one embodiment, the first shell channel inlet 104 and / or the second shell channel inlet 130 include two or more shell inlet subchannels 126 having different fluid reservoirs, input orifices, and control valves that converge to form a single shell channel outlet 106, 132 and / or fluid dispensing orifice. In an exemplary embodiment, a softer hydrogel material flowing through one shell inlet subchannel 126 can be switched to a harder hydrogel material flowing through a second shell inlet subchannel to reinforce the fiber if desired. In another exemplary embodiment, a first cell-containing material in one shell inlet subchannel 126 can be switched to a second cell-containing material in a second shell inlet subchannel 126 to produce a range of cell types along the length of the fiber.

[0023] In one embodiment, the first shell channel inlet 104 and / or the second shell channel inlet 130 include three or more shell inlet subchannels 126 having different fluid reservoirs, input orifices, and control valves that converge to form a single shell channel outlet 106, 132 and / or fluid dispensing orifice. In a preferred embodiment, one of the three shell inlet subchannels 126 includes a buffer solution input orifice and a control valve and is configured to dispense a buffer to facilitate displacement of the cross-linkable material within the dispensing channel 110.

[0024] In additional embodiments, the first shell channel inlet 104 and / or the second shell channel inlet 130 includes two or more sub-channels configured to deliver fluid to the first shell channel outlet 106 and / or the second shell channel outlet 132, each sub-channel converging at a separate fluid distribution orifice connecting the first shell channel inlet 104 and / or the second shell channel inlet 130 with the vertex 116 of the upper curved surface 114 of the first shell channel outlet 106 and / or the second shell channel outlet 132. In a preferred embodiment, the separate fluid distribution orifices are located on opposite sides of the first shell channel outlet 106 and / or the second shell channel outlet 132. In an exemplary embodiment, the upper curved surface 114 has a parabolic or elliptical shape.

[0025] In one embodiment, the first shell channel includes at least one fluid distribution orifice connecting the first shell channel inlet 104 with the vertex 116 of the upper curved surface 114 of the first shell channel outlet 106 so that the fluid is dispersed along the upper curved surface 114 and around the circumference of the first shell channel outlet 106, preferably wherein 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 subchannels 126 that converge at or near a single fluid distribution orifice. In another embodiment, the first shell channel includes at least two first shell inlet subchannels 126 that converge at or near two fluid distribution orifices, preferably located on opposite sides of the first shell channel outlet 106.

[0026] In another embodiment, the second shell channel 128 includes at least one fluid distribution orifice connecting the second shell channel inlet 130 with the vertex 116 of the upper curved surface 114 of the second shell channel outlet 132, such that the fluid is dispersed along the circumference of the upper curved surface 114 and the second shell channel outlet 132, preferably wherein 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 near a single 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 near two fluid distribution orifices, preferably located on opposite sides of the second shell channel outlet 132.

[0027] In some embodiments, the printhead further comprises a sheath flow channel 118 that converges with the distribution channel 110 at a sheath flow intersection point located between the fluid focusing chamber and the distal end of the distribution channel 110. In some embodiments, the sheath flow channel 118 comprises a plurality of sheath flow sub-channels that converge toward the distribution channel 110 via a sheath flow chamber 120. In a preferred embodiment, the sheath flow chamber 120 comprises a frustoconical shape configured to focus the fluid toward the distribution channel 110. In some embodiments, the sheath flow intersection point is located in the last / final downstream layer of the printhead. In some embodiments, the distribution channel 110 extends from the second-to-last layer of the printhead into the sheath flow chamber 120 in the final downstream layer.

[0028] In one embodiment, the minimum diameter of the frustum at the outlet of the fluid focusing chamber and the sheath fluid chamber 120 is the same and can be varied to adjust the total fiber diameter, for example, between about 0.01 mm and about 5 mm. In some embodiments, the printhead further comprises a dispensing orifice at the distal end of the dispensing channel 110. In some embodiments, the printhead further comprises an extension tip comprising a tube having an exterior configured to fit into a portion of the dispensing channel 110 and an interior surface configured to align with the dispensing channel 110 (defining a hollow space in the tube).

[0029] In one embodiment, the sheath flow channel 118 includes a sheath fluid input orifice and a control valve; preferably wherein the print head is configured to distribute the sheath fluid through the sheath flow channel 118. In some embodiments, the sheath fluid includes a chemical crosslinking agent. In some embodiments, the sheath fluid includes an aqueous solvent.

[0030] In another aspect, the present invention provides a printhead comprising a plurality of stacked, preferably bonded, layers forming a plurality of fluid channels, the printhead comprising: a core channel; a plurality of shell channels; and a fluid focusing chamber converging toward a distribution channel 110; wherein the core channel is in fluid communication with the fluid focusing chamber and extends longitudinally through a central region of the fluid focusing chamber and is aligned with the distribution channel 110; wherein the plurality of shell channels are concentrically disposed about the core channel in the same layer of the printhead and in fluid communication with the fluid focusing chamber, wherein the inner shell channels extend into the fluid focusing chamber for a longer length than the outer shell channels, and wherein the core channel extends into the fluid focusing chamber for a longer length than any of the shell channels; and a sheath channel 118 converging with the distribution channel 110 at a sheath fluid intersection point located between the fluid focusing chamber and a distal end of the distribution channel 110.

[0031] In some embodiments, the printhead further comprises a plurality of fluid distribution orifices configured to distribute fluid around the circumference of the plurality of shell channels, wherein the plurality of fluid distribution orifices individually connect a corresponding shell channel inlet 122 with a vertex 116 of an upper curved surface 114 of a corresponding shell channel outlet in the plurality of shell channels, preferably wherein the upper curved surface 114 of the second shell channel outlet 132 has a parabolic or elliptical shape. In an exemplary embodiment, at least one shell channel in the plurality of shell channels has a gradient width that increases with increasing longitudinal depth into the housing.

[0032] In some embodiments, the printhead further comprises a third, fourth, fifth, and / or sixth shell channel having at least one inlet and an outlet, wherein each of the third, fourth, fifth, and / or sixth shell channel outlets is concentrically disposed about an immediately preceding shell channel outlet in a multi-channel housing in the same layer of the printhead and is in fluid communication with a fluid focusing chamber. In some embodiments, the fluid focusing chamber is located in a separate layer of the printhead, and the shell channel outlets extend from a preceding layer of the printhead into a second fluid focusing 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 housing than the immediately preceding shell channel outlet, and the core channel extends further into the multi-channel housing than the first shell channel.

[0033] In an alternative embodiment, the printhead further comprises: a third, fourth, fifth and / or sixth shell channel, each shell channel having at least one inlet and outlet, and a third, fourth, fifth and / or sixth multi-channel housing located between the second fluid focusing chamber 136 and the distal end of the distribution channel 110, wherein the third, fourth, fifth and / or sixth multi-channel housing comprises the distribution channel 110, the third, fourth, fifth and / or sixth shell channel outlet, and the third, fourth, fifth and / or sixth fluid focusing chamber; wherein the distribution channel 110 is disposed in a central region of the respective multi-channel housing, with the distribution channel 110 disposed adjacent the distribution channel 110. The inlet of the corresponding fluid focusing chamber is in fluid communication and extends to a first vertical depth into the multi-channel housing, wherein the outlet of the third, fourth, fifth, and / or sixth shell channel is concentrically disposed about the distribution channel 110 and in fluid communication with the inlet of the corresponding fluid focusing chamber and extends to a second vertical depth into the corresponding multi-channel housing; and wherein the third, fourth, fifth, and / or sixth fluid focusing chambers converge toward the distribution channel 110, preferably wherein the third, fourth, fifth, and / or sixth fluid focusing chambers comprise a frusto-conical shape configured to focus fluid toward the distribution channel 110. In some embodiments, the third, fourth, fifth, and / or sixth multi-channel housing is located on successive layers of the printhead. In some embodiments, the shell channel outlets can extend from a previous layer of the printhead into corresponding fluid focusing chambers in an adjacent downstream layer of the printhead.

[0034] In a preferred embodiment, the present invention demonstrates a printhead comprising a plurality of stacked layers forming a plurality of fluid channels, the printhead comprising: a core channel comprising at least two core inlet sub-channels having different fluid reservoirs, input orifices, and control valves that converge to form a single core channel outlet 102 in fluid communication with a first fluid focusing chamber 112; a first shell channel comprising at least two shell inlet sub-channels 126 having different fluid reservoirs, input orifices, and control valves that converge to form a single shell channel outlet in fluid communication with a second fluid focusing chamber 136; a distribution channel 110; wherein the fluid focusing chamber converges toward the distribution channel, preferably wherein the fluid focusing chamber comprises a frusto-conical shape configured to focus fluid toward the distribution channel 110; and a sheath channel 118 that converges with the distribution channel 110 at a sheath fluid intersection point located between the second fluid focusing intersection point and a distal end of the distribution channel 110. In some embodiments, at least two wick inlet subchannels converge at or near the wick channel outlet 102, and preferably converge in the same or immediately preceding layer of the printhead as the wick 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 including a buffer solution.

[0035] In some embodiments, the core channel further comprises at least one fluid distribution orifice configured to distribute fluid around the circumference of the core channel outlet 102; preferably, wherein the at least one fluid distribution orifice connects the converging core channel inlet with the vertex 116 of the upper curved surface 114 of the core channel outlet 102; more preferably, wherein the upper curved surface 114 has a parabolic or elliptical shape.

[0036] Aspects of the present invention include a system for producing a fibrous structure, the system comprising a printhead, the printhead comprising: a core channel having an inlet and an outlet; a first shell channel, the first shell channel having an inlet and an outlet; a multi-channel housing; and a distribution channel 110, wherein the multi-channel housing comprises the core channel outlet 102, the first shell channel outlet 106, and a fluid focusing chamber; wherein the core channel outlet 102 is disposed in a central region of the multi-channel housing, is in fluid communication with the inlet of the fluid focusing chamber, and extends to a first vertical depth into the multi-channel housing, preferably wherein the core channel outlet 102 extends to the first vertical depth into the fluid focusing chamber, aligned with the distribution channel 110; wherein the first shell channel an outlet 106 concentrically disposed about the core channel, in fluid communication with an inlet fluid of the fluid focusing chamber, and extending to a second vertical depth into the multi-channel housing; and wherein the fluid focusing chamber converges toward the distribution channel 110, preferably wherein the fluid focusing chamber comprises a frusto-conical shape configured to focus the fluid toward the distribution channel 110; a sheath flow channel 118 converging with the distribution channel 110 at a sheath fluid intersection point located between a first fluid focusing intersection point and a distal end of the distribution channel 110; a receiving surface for receiving a first layer of material dispensed from the printhead; and a positioning assembly for positioning the distribution orifice of the printhead in 3D space, wherein the positioning assembly is operably coupled to the printhead.

[0037] In some embodiments, the system further comprises a programmable control processor for controlling the positioning assembly and for controlling the flow rate of one or more fluids through the printhead. In some embodiments, the system further comprises a fluid removal assembly, which is configured to remove excess fluid distributed from the printhead. In some embodiments, the fluid removal assembly comprises a porous membrane configured to allow excess fluid to pass through. In some embodiments, the fluid removal assembly comprises an absorbent material. In some embodiments, the fluid removal assembly comprises a vacuum configured to suck excess fluid. In some embodiments, vacuum is applied below the receiving surface. In some embodiments, vacuum is applied above the receiving surface. In some embodiments, vacuum is applied by one or more vacuum channels on the printhead. In some embodiments, one or more vacuum channels are positioned near the distribution orifice on the printhead.

[0038] In some embodiments, the system further comprises a pressure control assembly 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 solution. In some embodiments, the sheath solution comprises a cross-linking solution configured to solidify the input material. In some embodiments, the cross-linking solution comprises divalent cations. In some embodiments, the divalent cations are Ca++. In some embodiments, the fluid reservoir comprises a buffer solution. In some embodiments, the buffer solution is miscible with the input material. In some embodiments, the fluid reservoir comprises an 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 a 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.

[0039] In some embodiments, the system further comprises a printhead comprising at least two shell inlet subchannels 126, and / or a first shell channel comprising at least two shell subchannels connected to a fluid reservoir comprising different input materials, and the method comprises generating a core-shell fiber structure comprising the first input material and the 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 cured 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 cured fiber structure comprising different shell materials along the length of the continuous fiber structure.

[0040] 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 cross-linking assembly. In some embodiments, the cross-linking assembly comprises a UV lamp. In some embodiments, the cross-linking assembly is located near the dispensing orifice.

[0041] Aspects of the present invention include a method for producing a solidified fiber structure, the method comprising: providing a system for producing a fiber structure, the system comprising: a printhead comprising a plurality of stacked and preferably bonded layers forming a plurality of fluid channels, the printhead comprising a core channel having an inlet and an outlet, a first shell channel having an inlet and an outlet, a first multi-channel housing 108, and a distribution channel 110, wherein the multi-channel housing 108 comprises the core channel outlet 102, the first shell channel outlet 106, and a first fluid focusing chamber 112; wherein the core channel outlet 102 is disposed in a central region of the multi-channel housing 108, is in fluid communication with the inlet of the first fluid focusing chamber 112, and extends to a first vertical depth into the multi-channel housing 108, preferably wherein the core channel outlet 102 extends to the first vertical depth into the fluid focusing chamber, aligned with the distribution channel 110; wherein the first shell channel outlet 106 is concentrically disposed about the core channel, is in fluid communication with the inlet of the fluid focusing chamber, and extends to a second vertical depth into the multi-channel housing 108; and wherein the fluid focusing chamber converges toward the distribution channel 110 , preferably wherein the fluid focusing chamber comprises a truncated conical shape configured to focus the fluid toward the distribution channel 110; a sheath flow channel 118, which converges with the distribution channel 110 at a sheath fluid intersection located between a first fluid focusing intersection and the distal end of the distribution channel 110; a receiving surface for receiving a first layer of material dispensed from the print head; a positioning assembly for positioning the distribution orifice of the print head in 3D space, wherein the positioning assembly is operably coupled to the print head; a programmable control processor for controlling the positioning assembly and for controlling the flow rate of one or more fluids through the print head; a first fluid reservoir, the first fluid reservoir comprising a first input material; a second fluid reservoir, the second fluid reservoir comprising a buffer solution; and a third fluid reservoir, the third fluid reservoir comprising a sheath solution, wherein the sheath solution comprises a cross-linking solution; wherein the fluid reservoir is in fluid communication with the print head; passing the first input material through the distribution channel 110; cross-linking the first input material with the cross-linking assembly to produce a cured fiber structure; and dispensing the cured fiber structure from the distribution orifice of the print head.

[0042] In a preferred embodiment, the method includes simultaneously dispensing a buffer solution and / or sheath fluid through the core channel, one or more input materials through one or more shell channels, and sheath fluid through the sheath flow channel 118 to form a hollow core in the printed fiber.

[0043] 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 to solidify the outer surface of the crosslinkable material flow in the distribution channel 110.

[0044] In some embodiments, the non-crosslinkable material in the core channel comprises a chemical crosslinker and the sheath fluid in the sheath flow channel 118 comprises an aqueous solvent, and contact occurs at the first fluid focusing intersection to solidify the inner surface of the crosslinkable material flow in the distribution channel 110.

[0045] In some embodiments, the non-crosslinkable material in the core channel includes a chemical crosslinker and the sheath fluid in the sheath flow channel 118 includes a chemical crosslinker, and contact occurs at the first fluid focusing intersection to solidify the inner surface of the crosslinkable material flow, and contact occurs at the sheath fluid intersection to solidify the outer surface of the crosslinkable material flow in the distribution channel 110.

[0046] In some embodiments, a method further comprises: encoding a programmable control processor with a planar structure to be printed; and depositing a first layer of a cured fiber structure on a receiving surface to print the planar structure.

[0047] In some embodiments, a method further comprises: encoding a programmable control processor with a 3D structure to be printed; and depositing subsequent layers of a solidified fiber structure on top of the planar structure to print the 3D structure.

[0048] In another embodiment, the present invention provides a method for producing a continuous solidified fibrous structure having a core and / or sheath component that varies along the length of the fiber, the method comprising: providing a system for producing a fibrous structure, the system comprising: a printhead comprising a core channel, the core channel comprising at least two core inlet subchannels having different fluid reservoirs, input orifices, and control valves that converge to form a single core channel outlet 102 in fluid communication with a first fluid focusing chamber 112; and a first shell channel comprising at least two shell inlet subchannels 126, the shell inlet subchannels having distinct fluid reservoirs, input orifices, and control valves that converge to form a single shell channel outlet in fluid communication with a second fluid focusing chamber 136; and a distribution channel 110; wherein the fluid focusing chambers converge toward the distribution channel 110, preferably wherein the fluid focusing chambers comprise a frusto-conical shape configured to focus fluid toward the distribution channel 110; a sheath flow channel 118 that converges with the distribution channel 110 at a sheath fluid intersection point located between the second fluid focusing intersection point and a distal end of the distribution channel 110; a receiving surface for receiving fluid dispensed from the printhead a first layer of material; a positioning assembly for positioning a dispensing orifice of the printhead in 3D space, wherein the positioning assembly is operably coupled to the printhead; a programmable control processor for controlling the positioning assembly and for controlling a flow rate of one or more fluids through the printhead; a first fluid reservoir, the first fluid reservoir comprising a first input material connected to the first core inlet subchannel; a second fluid reservoir, the second fluid reservoir comprising a second input material connected to the second core inlet subchannel; a third fluid reservoir, the third fluid reservoir comprising a third input material connected to the first shell inlet subchannel 126; a fourth A fluid reservoir comprising a fourth input material connected to the second shell inlet subchannel 126; a fifth fluid reservoir comprising a sheath solution connected to the sheath flow channel 118, wherein the sheath solution comprises a crosslinking solution; wherein the fluid reservoir is in fluid communication with the printhead; alternately passing the first or second input material through the dispensing channel 110; and simultaneously alternately passing the third or fourth input material through the dispensing channel 110, crosslinking the first, second, third, and / or fourth input materials with the crosslinking component to produce a cured fiber structure; and dispensing the cured fiber structure from the dispensing orifice of the printhead. In some embodiments, the first and / or second input material comprises a non-crosslinkable material.

[0049] In another embodiment, the first shell channel includes three shell inlet subchannels, and a sixth fluid reservoir including a buffer solution is connected to the third shell inlet subchannel 126, and the method includes passing the buffer solution through the distribution channel 110 to displace the crosslinkable material and terminate the fibers.

[0050] In another aspect, bioprinted tissue fibers produced by the subject methods are also contemplated to have variable core and sheath materials throughout the length of the fiber.

[0051] The present invention also successfully addresses the technical challenges of manufacturing synthetic perfusable hollow tissue fibers that are capable of being attached to an external perfusion system without breaking, as detailed and illustrated in Example 1 herein. In one aspect, the present invention provides a bioprinted tissue fiber comprising: an inner lumen; a continuous inner shell surrounding the inner lumen, the inner shell comprising a reinforcing hydrogel material at the distal and proximal ends of the fiber; and a biocompatible hydrogel material located therebetween. The biocompatible hydrogel material preferably comprises at least one biological material, such as living cells, while the reinforcing hydrogel material does not contain cells. In another embodiment, the fiber further comprises a second outer continuous shell comprising the reinforcing hydrogel material.

[0052] In some embodiments, the enhanced hydrogel material is selected from, for example, alginate, chitosan, acrylated PEG, including but not limited to PEGDA, PEGTA, polyvinyl alcohol (PVA), PCL, PLGA. In some embodiments, the biocompatible hydrogel material is selected from, for example, alginate, chitosan, acrylated PEG, ECM factors, including collagen, laminin, fibronectin, vitronectin, fibrin / fibrinogen, decellularized tissue ECM, hyaluronic acid, gelatin, and methacrylated gelatin. In an exemplary embodiment, the enhanced hydrogel material includes a higher concentration of alginate material, for example, 3.5wt% to 4.5wt%, preferably 3.8wt% to 4.2wt%, more preferably about 4wt%, and the biocompatible hydrogel material includes a lower concentration of alginate material, for example, 1.0wt% to 1.5wt%, preferably 1.2wt% to 1.4wt%, more preferably about 1.3wt%.

[0053] In some embodiments, at least one biomaterial comprises living cells, such as cells from endocrine and exocrine glands, including pancreas (α, β, δ, ε, γ), liver (hepatocytes, Kupffer cells, stellate cells, sinusoidal cells), thyroid (follicular cells), pineal gland (pinealocytes), pituitary gland (growth hormone, prolactin, gonadotropin, adrenocorticotropic hormone, and thyrotropin), thymus (thymocytes, thymic epithelial cells, thymic stromal cells), adrenal gland (cortical cells, chromaffin cells), ovary (granulosa cells), testis (interstitial cells), and gastrointestinal tract (enteroendocrine cells - intestine, stomach, pancreas). In preferred embodiments, at least one biomaterial comprises a cell population that expresses / secretes a bioactive agent, such as insulin, glucagon, growth hormone, pancreatic polypeptide, angiogenic factors, growth factors, hormones, antibodies, enzymes, proteins, exosomes, etc.

[0054] Aspects of the present invention also include a method for producing perfusable hollow tissue fibers, the method comprising: providing a printhead according to the present invention, the printhead comprising a first shell channel, the first shell channel comprising at least two shell inlet subchannels 126, the shell inlet subchannels having different fluid reservoirs, input orifices, and control valves; dispensing sheath fluid through the core channel, reinforcing hydrogel material through the first shell inlet subchannel 126, a biocompatible hydrogel material comprising one or more biomaterials through the second shell inlet subchannel 126, and sheath fluid through the sheath flow channel 118; and transitioning between the reinforcing hydrogel material and the biocompatible hydrogel material along the length of the printed fiber. In this way, the reinforcing material can be incorporated into the end of the perfusable fiber to enable attachment to an external perfusion system, such as by needle insertion. In another embodiment, the printhead further comprises a second shell channel 128 as described herein, and the method further comprises dispensing the same or different reinforcing hydrogel material through the second shell channel 128 to produce a concentric second shell surrounding the first shell material and further reinforcing the fiber and preventing breakage along the entire length of the fiber.

[0055] The present invention also successfully addresses the technical challenges of manufacturing synthetic perfusable hollow tissue fibers that are capable of being attached to an external perfusion system without breaking, as detailed and illustrated in Example 1 herein. In one aspect, the present invention provides a bioprinted tissue fiber comprising: an inner lumen; a continuous inner shell surrounding the inner lumen, the inner shell comprising a reinforcing hydrogel material at the distal and proximal ends of the fiber; and a biocompatible hydrogel material located therebetween. The biocompatible hydrogel material preferably comprises at least one biological material, such as living cells, while the reinforcing hydrogel material does not contain cells. In another embodiment, the fiber further comprises a second outer continuous shell comprising the reinforcing hydrogel material. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 is an illustration of a concentric shell printhead design of the present invention having a single fluid distribution orifice.

[0057] Figure 2 Side views and close-up detail views are provided of the multi-channel housing and sheath flow chamber in the concentric shell printhead design of the present invention with a single fluid distribution orifice.

[0058] Figure 3 Key components of the microfluidic pathway in the present concentric shell printhead design with a single fluid distribution orifice are shown and identified.

[0059] Figure 4Ais a graphical representation of the flow pattern through a single fluid distribution orifice in the concentric shell printhead design of the present invention. Figure 4B is a graphical representation of the flow pattern through a single fluid dispensing orifice in the multi-channel housing and sheath flow chamber of the concentric shell printhead design of the present invention.

[0060] Figure 5 Side views and close-up detail views are provided of the multi-channel housing and sheath flow chamber in the multi-shell concentric printhead design of the present invention having two fluid input orifices.

[0061] Figure 6 Key components of the microfluidic pathway in the multi-shell concentric printhead design of the present invention having two fluid distribution orifices are shown and identified.

[0062] Figure 7A is a diagrammatic representation of the flow pattern through two fluid distribution orifices in a multi-shell printhead design of the present invention having two fluid distribution orifices, and Figure 7B is a graphical representation of the flow pattern through two fluid distribution orifices in the multi-channel housing and sheath flow chamber of the multi-shell printhead design of the present invention.

[0063] Figure 8A and Figure 8B Illustrations and close-up detail views are provided of an embodiment of the present invention comprising two distinct shell inlet subchannels converging at a single fluid distribution orifice.

[0064] Figures 9A to 9C Transparent (10A), top (10B), and exploded layer (10D) views are provided of a printhead design according to the present invention comprising three distinct shell inlet subchannels converging at a single fluid distribution orifice.

[0065] FIG. 10A to FIG. 10C Transparent views (10A), top views (10B), and exploded layer views (10C) of a printhead design according to the present invention are provided, the printhead design including a second shell channel, a second multi-channel housing, and a second fluid focusing chamber in a different layer of the printhead than the first fluid focusing chamber.

[0066] Figure 11 A transparent diagram of a printhead design according to the present invention is provided, the printhead design including a second shell channel, a second multi-channel housing, and a second fluid focusing chamber overlapping the first fluid focusing chamber, wherein the second multi-channel housing overlaps the first fluid focusing chamber in the same layer of the printhead.

[0067] Figure 12 Various illustrations of perfusable hollow tissue fibers according to the present invention are provided.

[0068] Figure 13Additional illustrations of perfusable hollow tissue fibers according to the present invention are provided.

[0069] Figure 14 Various illustrations are provided of synthetic tissue fibers according to the present invention that include core and / or sheath compositions that vary along the length of the fiber.

[0070] Figure 15 Synthetic hollow tissue fibers according to the present invention are provided having a range of lumen and fiber diameters. DETAILED DESCRIPTION

[0071] Aspects of the invention include systems and methods for producing core-shell fiber structures including hollow core fibers and multi-shell fibers and for producing three-dimensional (3D) structures from digital files. In some embodiments, the printed fibers include living cells.

[0072] definition:

[0073] For the purpose of interpreting this specification, the following definitions will apply, and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event of any conflict between any definition set forth and any document incorporated herein by reference, the definition set forth below shall prevail.

[0074] 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 solution is configured to displace the input material from a location within the distribution channel 110 (e.g., from the proximal end of the distribution channel 110). In some embodiments, the displacement is an instantaneous displacement that occurs in less than about one second, such as about 900, 800, 700, 600, 500, 400, 300, 200, or 100 milliseconds or less.

[0075] As used herein, the term "miscible" refers to the ability of two different liquids to form a homogeneous mixture when mixed.

[0076] As used herein, the term "mass flow rate" refers to the mass of a substance 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.

[0077] As used herein, the term "cured" refers to a solid or semi-solid material that maintains its shape fidelity and structural integrity when deposited. As used herein, the term "shape fidelity" refers to the ability of a material to maintain its three-dimensional shape without significant diffusion. In some embodiments, a cured material is a material having the ability to maintain its three-dimensional shape for a period of about 30 seconds or longer, such as about 1, 10 or 30 minutes or longer, such as about 1, 10, 24 or 48 hours or longer. As used herein, the term "structural integrity" refers to the ability of a material to hold together under a load including its own weight while resisting breakage or bending.

[0078] In some embodiments, the solidified composition is a composition having an elastic modulus 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 elastic modulus ranges include about 5, 10, 15, 20, 25 or 50 Pa to about 80, 100, 120 or 140 kPa. According to the present invention, the elastic modulus of the input material can be advantageously changed according to the intended function of the input material. In some embodiments, a lower elastic modulus is used to support cell growth and migration, while in other embodiments, a much higher elastic modulus can be used.

[0079] As used herein, the term "natural alginate polymer" refers to alginate polymer that has been isolated and purified from one or more natural sources (eg, one or more brown seaweeds or seaweeds).

[0080] As used herein, the term "depolymerization" refers to the breaking of polymer chains into monomers or other smaller units.

[0081] As used herein, the term "hydrogel" refers to a composition comprising a network or lattice of water and hydrophilic polymer chains.

[0082] As used herein, the term "sheath fluid" or "sheath solution" refers to a fluid that is used, at least in part, to encapsulate or "wrap" a material as it passes through a fluidic 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 crosslinking agent. Non-limiting examples of crosslinking agents include divalent cations (e.g., Ca 2+ 、Ba 2+ 、Sr 2+ etc.), thrombin and pH modifying chemicals such as sodium bicarbonate.

[0083] As used herein, the term "excess sheath fluid" refers to a portion of the sheath fluid dispensed from the 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, excess sheath fluid can be used to lubricate the passage of a material (e.g., a hydrogel) through the dispensing channel 110 in the print head and through the passage of the dispensing orifice. Once dispensed from the dispensing orifice, excess sheath fluid may flow from the surface of the dispensed material layer and onto a receiving surface where it may collect or pool.

[0084] 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.

[0085] As used herein, the term "convergence angle" refers to the angle formed between two converging fluid channels.

[0086] Printhead:

[0087] Aspects of the present invention include print heads that can be used to produce one or more core-shell fiber structures, the core-shell fiber structures comprising multi-shell fibers and / or hollow fibers. A print head according to an embodiment of the present invention includes a plurality of stacked layers forming a plurality of interconnected fluid channels that flow vertically through the layers, the layers preferably being combined together to form a common shell or outer shell, and configured to produce a core-shell fiber structure comprising one or more input materials. In some embodiments, the print head is configured to produce a cured hollow fiber structure. In some embodiments, the print head is configured to produce a cured hollow fiber structure comprising living cells.

[0088] In some embodiments, the printhead includes a distribution channel 110 having a distal end and a proximal end. Distribution channels according to embodiments of the present invention can have a channel length ranging from about 1 mm to about 100 mm, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or about 95 mm. Distribution channels according to embodiments of the present invention can have a width or diameter ranging from about 10 μm to about 5 mm, for example, 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. The distribution channel according to an embodiment of the present invention can have a depth ranging from about 10 μm to about 5 mm, for example, 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. The distribution channel according to an embodiment of the present invention can have any suitable cross-sectional shape, for example, circular, oval, square or rectangular cross-sectional shape.

[0089] In some embodiments, the distribution channel 110 includes a distribution orifice. In some embodiments, the distribution orifice is located at the distal end of the distribution channel 110. The distribution orifice according to embodiments of the present invention can have a diameter ranging from about 10 μm to about 5 mm, for example, 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. The distribution orifice according to embodiments of the present invention can have any suitable cross-sectional shape, for example, a circular, oval, square or rectangular cross-sectional shape.

[0090] In some embodiments, the print head also includes an extension tip, which includes an orifice for dispensing material from the print head. This extension tip contributes to the precise distribution of material and the deposition of material in a confined area, such as a hole in a multi-well plate (e.g., a standard microtiter plate, a multi-well plate, or a microplate with 6, 24, 96 or more holes) or a culture dish. In some embodiments, the extension tip includes a tube (e.g., made of plastic, glass, or metal) having an exterior configured to fit into a portion of distribution channel 110 and an inner surface configured to align with the distribution channel (defining the hollow space in the tube). The extension tip can be inserted into the distribution channel 110, thereby extending the length of the distribution channel 110, which facilitates the deposition of material dispensed from the orifice in the extension tip into a confined space such as a well plate insert or a culture dish.

[0091] The print head according to an embodiment of the present invention includes one or more wick channels. In certain embodiments, one or more wick channels converge with the distribution channel 110 at the proximal end of the distribution channel 110. In some embodiments, the wick channel converges with the distribution channel 110 at a certain convergence angle, and the convergence angle ranges from about 0 to about 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. The wick channel according to an embodiment of the present invention can have any suitable channel length. In some embodiments, the core channel has a channel length ranging 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. The core channel according to embodiments of the present invention can have a width or diameter ranging from about 10 μm to about 5 mm, for example, 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. Material channels according to embodiments of the present invention may have a depth ranging from about 10 μm to about 5 mm, such as about 25, 50, 75, or 100 μm, or such as 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.

[0092] In some embodiments, the printhead includes at least two wick channels having the same or different fluid reservoirs, input orifices, and control valves that converge to form a single wick channel outlet 102 in fluid communication with the fluid focusing chamber. In a preferred embodiment, the at least two wick channels converge at or near the wick channel outlet 102, the multi-channel housing 108, and / or the fluid dispensing orifice described further herein, for example, within about 100 μm to about 50 mm, thereby reducing travel distance and bringing the material transition point closer to the solidification point, which the inventors have determined can prevent smearing between material transitions within the printed fiber. In a particularly preferred embodiment, the at least two sub-channels converge in the immediately preceding layer, or in the same layer of the printhead as the wick channel outlet 102, the multi-channel housing, and / or the fluid dispensing orifice. In some embodiments, the channel length between the location where the subchannels converge and the core channel outlet 102, the multichannel housing 108 and / or the fluid distribution orifice ranges from about 100 μm to about 50 mm, for example, 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 includes a plurality of core subchannels ranging from 3 to 10, for example, 4, 5, 6, 7, 8 or 9 subchannels. The core channel according to an embodiment of the present invention can have any suitable cross-sectional shape, for example, a circular, elliptical, square or rectangular cross-sectional shape. In some embodiments, the print head is configured to distribute non-crosslinkable material through the core channel.

[0093] A printhead according to an embodiment of the present invention includes: a wick channel having an inlet and an outlet; a first shell channel having an inlet and an outlet; a multi-channel housing 108; and a distribution channel 110, wherein the multi-channel housing 108 includes the wick channel outlet 102, the first shell channel outlet 106, and a fluid focusing chamber; wherein the wick channel outlet 102 is disposed in a central region of the multi-channel housing 108, is in fluid communication with the inlet of the fluid focusing chamber, and extends to a first vertical depth into the multi-channel housing 108, preferably wherein the wick channel outlet 102 extends to a first vertical depth into the fluid focusing chamber, aligned with the distribution channel 110; wherein the first shell channel outlet 106 is concentrically disposed about the wick channel, is in fluid communication with the inlet of the fluid focusing chamber, and extends to a second vertical depth into the multi-channel housing 108; and wherein the fluid focusing chamber converges toward the distribution channel 110, preferably wherein the fluid focusing chamber includes a frusto-conical shape configured to focus fluid toward the distribution channel 110. In some embodiments, first shell channel outlet 106 has a gradient width that increases with depth into multi-channel housing 108. In an exemplary embodiment, first shell channel outlet 106 comprises a hollow cylinder having an axis of rotation that does not intersect core channel outlet 102.

[0094] In some embodiments, core channel outlet 102 extends through a majority of the length of multichannel housing 108. In preferred embodiments, the first vertical depth is greater than the second vertical depth, such that core channel outlet 102 extends further into multichannel housing 108 and / or first fluid focusing chamber 112 than first shell channel outlet 106. In alternative embodiments, the second vertical depth is greater than the first vertical depth, such that first shell channel outlet 106 extends further into multichannel housing 108 than core channel outlet 102.

[0095] In some embodiments, the printhead further includes a second shell channel 128 having an inlet and an outlet, wherein the second shell channel outlet 132 and / or the inlet 130 are concentrically disposed about the first shell channel outlet 106 in the multi-channel housing 108 in the same layer of the printhead and in fluid communication with the fluid focusing chamber. In a preferred embodiment, the first shell channel outlet 106 extends further into the multi-channel housing 108 than the second shell channel outlet 132. In some embodiments, the second shell channel inlet 130 can be adjacent to the first shell channel outlet 106 in the same layer of the printhead and can be in fluid communication with the first fluid focusing chamber 112.

[0096] In an alternative embodiment, the printhead further comprises: a second shell channel having at least one inlet and an outlet; and a second multi-channel housing positioned between the first fluid focusing chamber and a distal end of the distribution channel, wherein the second multi-channel housing comprises the distribution channel, the second shell channel outlet, and the second fluid focusing chamber; wherein the distribution channel is positioned in a central region of the second multi-channel housing, is in fluid communication with the inlet of the second fluid focusing chamber, and extends to a first vertical depth into the multi-channel housing, preferably wherein the second shell channel outlet is concentrically positioned about the distribution channel, is in fluid communication with the inlet of the second fluid focusing chamber, and extends to a second vertical depth into the multi-channel housing; and wherein the second fluid focusing chamber converges toward the distribution channel, preferably wherein the second fluid focusing chamber comprises a frusto-conical shape configured to focus fluid toward the distribution channel. In some embodiments, the first and second multi-channel housings, or portions thereof, are positioned in the same layer of the printhead, for example, as Figure 11 As shown, the second multichannel housing can overlap the first fluid focusing chamber. In some embodiments, the first and second multichannel housings are located in consecutive layers of the printhead, e.g., as shown in FIG. Figure 10A In some embodiments, the second fluid focusing chamber is located in a separate layer of the printhead, and the second shell channel outlet extends from a previous layer of the printhead to the second fluid focusing chamber in an adjacent downstream layer.

[0097] The core and shell channels according to embodiments of the present 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, 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. The core and shell channels according to embodiments of the present invention can have a width or diameter ranging from about 10 μm to about 5 mm, for example, 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 present invention can have a depth ranging from about 10 μm to about 5 mm, such as about 25, 50, 75, or 100 μm, or such as 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 present invention can have any suitable cross-sectional shape, such as a circular, oval, square, or rectangular cross-sectional shape.

[0098] 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 circumference of the first shell channel outlet 106 and / or the second shell channel outlet 132. Preferably, the fluid distribution orifice connects the first shell channel inlet 104 and / or the second shell channel inlet 130 to the vertex 116 of the upper curved surface 114 of the first shell channel outlet 106 and / or the second shell channel outlet 132. In some embodiments, the first and / or second shell channel comprises at least two shell sub-channels that can converge at a single fluid distribution orifice or lead to separate fluid distribution orifices. The shell sub-channels can be fluidically connected to the same fluid reservoir, input orifice, and control valve, or fluidically connected to separate fluid reservoirs, input orifices, and control valves. In some embodiments, the printhead comprises a plurality of shell sub-channels ranging from 3 to 10, for example, 4, 5, 6, 7, 8, or 9 shell sub-channels. Shell channels and sub-channels according to embodiments of the present invention may have any suitable cross-sectional shape, such as circular, oval, square, or rectangular cross-sectional shapes.

[0099] In additional embodiments, the first shell channel inlet 104 and / or the second shell channel inlet 130 includes two or more sub-channels configured to deliver fluid to the first shell channel outlet 106 and / or the second shell channel outlet 132, each sub-channel including a separate fluid distribution orifice connecting the first shell channel inlet 104 and / or the second shell channel inlet 130 with the vertex 116 of the upper curved surface 114 of the first shell channel outlet 106 and / or the second shell channel outlet 132, preferably wherein the upper curved surface 114 of the first shell channel outlet 106 and / or the second shell channel outlet 132 has a parabolic or elliptical shape. In a preferred embodiment, the separate fluid input orifices are located on opposite sides of the first shell channel outlet 106 and / or the second shell channel outlet 132. In an exemplary embodiment, the upper curved surface 114 has a parabolic or elliptical shape.

[0100] In one embodiment, the first shell channel includes at least one fluid distribution orifice connecting the first shell channel inlet 104 with the apex 116 of the upper curved surface 114 of the first shell channel outlet 106, such that the fluid is dispersed along the upper curved surface 114 and around the circumference of the first shell channel outlet 106. In another 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 with the apex 116 of the upper curved surface 114 of the second shell channel outlet 132, such that the fluid is dispersed along the upper curved surface 114 and the circumference of the second shell channel outlet 132. In another embodiment, the second shell channel 128 includes two fluid distribution orifices located on opposite sides of the second shell channel outlet 132.

[0101] 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 distribution channel 110 at a sheath fluid intersection point located between the first fluid focusing intersection point and the distal end of the distribution channel 110. In some embodiments, the sheath flow channel 118 converges with the distribution channel 110 at a convergence angle ranging from about 0 to about 180 degrees, such as 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 the distribution channel 110 and the sheath fluid intersection point 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 the distribution channel 110 and the sheath fluid intersection point 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.

[0102] In some embodiments, the sheath flow channel 118 has a channel length ranging 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. In some embodiments, the sheath flow channel 118 has a channel length ranging from about 100 μm to about 5 mm, for example, 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. The sheath flow channel according to an embodiment of the present invention can have a depth ranging from about 10 μm to about 5 mm, for example, 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. In some embodiments, the sheath flow channel 118 includes two or more sheath flow sub-channels. In some embodiments, the sheath flow channel 118 is bifurcated into a plurality of sheath flow sub-channels ranging from 3 to 10, for example, 4, 5, 6, 7, 8 or 9. In some embodiments, two or more sheath flow sub-channels converge with the distribution channel 110 at the sheath liquid intersection. The sheath flow channel according to an embodiment of the present invention can have any suitable cross-sectional shape, for example, a circular, elliptical, square or rectangular cross-sectional shape.

[0103] Fluid channels according to embodiments of the present invention typically include one or more input orifices through which fluid can be introduced into the channel, typically located in the first or topmost layer of the printhead stack. In some embodiments, the fluid channel includes a control valve configured to regulate fluid flow through the fluid channel, typically located in the second layer from the top of the printhead stack. In some embodiments, the channel length between the input orifice and the control valve 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. In some embodiments, the length of the channel between the control valve and the location where the channel converges with the distribution 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.

[0104] The print head according to an embodiment of the present invention can be made of any suitable material, including but not limited to plastics (such as polymeric materials), glass, metal, ceramics or any combination thereof. In a preferred embodiment, the print head is manufactured using known microfluidic forming technology (such as casting, imprinting or injection molding) and one or more formable polymers such as polydimethylsiloxane (PDMS), polycarbonate (PC), cycloolefin 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 technology can be used to manufacture the print head.

[0105] In some embodiments, the printhead comprises a material that is at least partially transparent to light (e.g., ultraviolet (UV) light). In some embodiments, the printhead is made entirely of a transparent material. In certain embodiments, a portion of the printhead surrounding or directly adjacent to the dispensing channel 110 comprises a material that is partially or completely transparent to light. Such printheads can be used in conjunction with input materials that are configured to crosslink with light energy (e.g., photocrosslinkable input materials).

[0106] Aspects of the present invention include a light module configured to expose a photocrosslinkable input material to electromagnetic radiation to crosslink the input material. Light modules according to embodiments of the present invention can be integrated into a printhead, or can be separate components of a printing system. In some embodiments, the light module exposes the input material to light while the input material is within the dispensing channel 110. In some embodiments, the light module exposes the input material to light after the input material is dispensed from the dispensing channel 110. In some embodiments, the printhead includes a plurality of light modules, wherein a first light module is configured to expose the input material to light while the input material is within the dispensing channel 110, and a second light module is configured to expose the input material to light after the input material is dispensed from the dispensing channel 110.

[0107] In some embodiments, the light module is tunable with respect to wavelength, intensity, exposure time, or any combination thereof. In some embodiments, the light module includes one or more optionally engaged attenuation filters, wherein the attenuation filter adjusts the light intensity when engaged. In some embodiments, the light module is configured to emit UV light, wherein the wavelength of the light emitted from the module ranges from about 10 nm to about 400 nm, for example, 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, as non-limiting examples, suitable UV light sources include UV lamps, UV fluorescent lamps, UV LEDs, UV lasers, or any combination thereof.

[0108] As described above, aspects of the present invention include a printhead comprising a distribution channel 110, wherein one or more material channels and optionally a buffer solution channel converge at a proximal end of the distribution channel 110. The printhead of the present invention is configured to dispense a buffer solution and / or a sheath fluid simultaneously with one or more cross-linkable materials to form a hollow core in the printed fiber. In some embodiments, the printhead is configured to maintain a constant mass flow rate through the distribution channel 110. In this manner, the printhead of the present invention is configured to facilitate a smooth and continuous flow of one or more input materials (or a mixture of one or more input materials) and a buffer solution and / or a sheath fluid through the distribution channel 110.

[0109] As described above, additional aspects of the present invention include a printhead comprising a distribution channel 110, wherein one or more sheath flow channels 118 converge with the distribution channel 110 at a sheath fluid intersection point located between a first fluid focusing intersection point and a distal end of the distribution channel 110. When using the printhead of the present invention, input material flowing through the distribution channel 110 can be cross-linked internally by sheath fluid flowing through the core channel, and externally by sheath fluid flowing through the sheath flow channels 118.

[0110] In a preferred embodiment, the present invention provides a printhead comprising a plurality of stacked, preferably bonded, layers forming a plurality of fluid channels, the printhead comprising: a core channel; a plurality of shell channels; and a fluid focusing chamber converging toward a distribution channel 110; wherein the core channel is in fluid communication with the fluid focusing chamber and extends longitudinally through a central region of the fluid focusing chamber and is aligned with the distribution channel 110; wherein the plurality of shell channels are concentrically disposed about the core channel in the same layer of the printhead and in fluid communication with the fluid focusing chamber, wherein the inner shell channels extend into the fluid focusing chamber for a greater length than the outer shell channels, and wherein the core channel extends into the fluid focusing chamber for a greater length than any of the shell channels; and a sheath channel 118 converging with the distribution channel 110 at a sheath fluid intersection point located between the fluid focusing chamber and a distal end of the distribution channel 110.

[0111] In some embodiments, the printhead further comprises a plurality of fluid distribution orifices configured to distribute fluid around the circumference of the plurality of shell channels, wherein the plurality of fluid distribution orifices individually connect a corresponding shell channel inlet 122 with a vertex 116 of an upper curved surface 114 of a corresponding shell channel outlet in the plurality of shell channels, preferably wherein the upper curved surface 114 of the shell channel outlet has a parabolic or elliptical shape. In an exemplary embodiment, at least one shell channel in the plurality of shell channels has a gradient width that increases with increasing longitudinal depth into the housing.

[0112] In some embodiments, the printhead further comprises a third, fourth, fifth, and / or sixth shell channel having an inlet and an outlet, wherein each of the third, fourth, fifth, and / or sixth shell channel outlets is concentrically disposed about an immediately preceding shell channel outlet in the multi-channel housing and is in fluid communication with the fluid focusing chamber. In a preferred embodiment, each of the third, fourth, fifth, and / or sixth shell channel outlets extends a shorter distance into the multi-channel housing than the immediately preceding shell channel outlet, and the core channel extends further into the multi-channel housing than the first shell channel.

[0113] In another preferred embodiment, the present invention provides a printhead comprising a plurality of stacked layers forming a plurality of fluid channels, the printhead comprising: a core channel comprising at least two core inlet subchannels having different fluid reservoirs, input orifices, and control valves that converge to form a single core channel outlet 102 in fluid communication with a first fluid focusing chamber 112, preferably wherein the at least two core inlet subchannels converge at or near the core channel outlet 102; a first shell channel comprising at least two shell inlet subchannels 126, the shell inlet subchannels having distinct fluid reservoirs, input orifices, and control valves that converge to form a single shell channel outlet in fluid communication with a second fluid focusing chamber 136; a distribution channel 110; wherein the fluid focusing chambers converge toward the distribution channel 110, preferably wherein the fluid focusing chambers comprise a frusto-conical shape configured to focus fluid toward the distribution channel 110; and a sheath flow channel 118 that converges with the distribution channel 110 at a sheath fluid intersection point located between the second fluid focusing intersection point and the distal end of the distribution channel 110. In another embodiment, the first shell channel comprises three shell inlet subchannels 126, one of which is connected to a fluid reservoir comprising a buffer solution.

[0114] In some embodiments, the core channel further comprises at least one fluid distribution orifice configured to distribute fluid around the circumference of the core channel outlet 102; preferably, wherein the at least one fluid distribution orifice connects the converging core channel inlet with the vertex 116 of the upper curved surface 114 of the core channel outlet 102; more preferably, wherein the upper curved surface 114 has a parabolic or elliptical shape.

[0115] Printing system:

[0116] Aspects of the present invention include a printing system and associated components configured to work together with the printhead of the present invention to perform the method of the present invention. In some embodiments, the printing system includes a single printhead, as described herein. In some embodiments, the printing system includes multiple printheads, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 separate printheads, as described herein. In some embodiments, the printhead is isolated from the printing system fluid so that all fluids related to the printing process remain isolated within the printhead and only contact the receiving surface (as described below) of the printing system during the printing process. In some embodiments, the printhead is configured to be operably coupled to the printing system without contacting the fluid involved in the printing process with the components of the printing system. In some embodiments, before, during and / or after the printing process, one or more printheads can be removed and / or one or more printheads can be added to the printing system. Therefore, in some embodiments, the printhead of the present invention is a modular component of the printing system of the present invention.

[0117] In some embodiments, the printing system includes a receiving surface on which the first layer of material dispensed from the dispensing orifice of the 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 porosity of the porous material is sufficient to allow fluid to pass therethrough. In some embodiments, the receiving surface is substantially planar, thereby providing a flat surface on which the first layer of dispensed material can be deposited. In some embodiments, the receiving surface has a topography corresponding to the three-dimensional structure to be printed, thereby facilitating the printing of a three-dimensional structure having a non-planar first layer.

[0118] In some embodiments, the receiving surface includes a vacuum assembly configured to apply suction from one or more vacuum sources to the receiving surface. In some embodiments, the receiving surface includes one or more vacuum channels configured to apply suction to the receiving surface. In some embodiments, the receiving surface including the vacuum assembly is configured to aspirate excess fluid from the receiving surface before, during, and / or after performing a printing process.

[0119] In some embodiments, the receiving surface is a non-cytotoxic surface onto which the printing system distributes one or more fibrous structures. In some embodiments, the printing system includes a printer table. In some embodiments, the receiving surface is a surface of the printer table. In some embodiments, the receiving surface is a component that is separate from the printer table but is fixed to the printer table or supported by the printer table. 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 accommodate the shape, size, texture, or geometry of the printed structure. In some embodiments, the receiving surface controls or influences the size, shape, texture, or geometry of the printed structure.

[0120] In some embodiments, the receiving surface comprises one or more modular components that are configured to be operably coupled to the printing system but detachable 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.

[0121] In some embodiments, the receiving surface is configured to be operably coupled to one or more different receiving containers. For example, in some embodiments, the receiving surface includes a circular portion sized to be operably coupled to a circular receiving container (e.g., a culture dish). In some embodiments, the receiving surface includes a square or rectangular portion sized to be operably coupled to a square or rectangular receiving container (e.g., a multi-well plate (e.g., a 6-well plate)). Receiving surfaces according to embodiments of the present invention can have any suitable size or geometry to accommodate a suitable receiving container.

[0122] In some embodiments, the printing system includes a temperature regulation assembly configured to regulate the temperature of the receiving surface. In some embodiments, the temperature regulation assembly regulates and / or maintains the temperature of the receiving surface at ambient temperature. In some embodiments, the temperature regulation assembly regulates and / or maintains the temperature of the printhead, printer stage, receiving surface, input material, and / or fluid (e.g., sheath solution and / or buffer solution).

[0123] In some embodiments, the temperature regulating assembly includes a heating element. In some embodiments, the temperature regulating assembly includes a heater. In some embodiments, the temperature regulating assembly includes a radiant heater, a convection heater, a conduction heater, a fan heater, a heat exchanger, or any combination thereof. In some embodiments, the temperature regulating assembly includes a cooling element. In some embodiments, the temperature regulating assembly includes a container for a coolant, a frozen liquid, ice, or any combination thereof. In some embodiments, the temperature regulating assembly includes a radiant cooler, a convection cooler, a conduction cooler, a fan cooler, or any combination thereof.

[0124] In some embodiments, the temperature regulating component is configured to regulate the temperature to a set point ranging from 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.

[0125] In some embodiments, the printing system achieves a specific geometry by moving the print head relative to a printer table or a receiving surface suitable for receiving printed material. In other embodiments, the printing system achieves a specific geometry by moving the printer table or the 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 biological safety cabinet (BSC)). In some embodiments, the printing system is configured to fully fit into a sterile environment.

[0126] In some embodiments, the receiving surface receives excess fluid (eg, excess sheath fluid and / or excess buffer solution) that is dispensed from the dispensing orifice and flows from the one or more layers of material dispensed from the dispensing orifice.

[0127] In some embodiments, the system includes a component for removing excess fluid (e.g., excess sheath fluid and / or excess buffer solution) from a receiving surface on which a fiber structure dispensed from an orifice of a print head is deposited, optionally from the surface of the dispensed fiber structure. During the printing process, excess fluid may collect or "pool" on the receiving surface or on the surface of the dispensed fiber structure. Such pooling may interfere with the deposition process. For example, the pooled sheath fluid may cause the dispensed fibers to slide from their intended position in the 3D structure being printed. Therefore, in some embodiments, the additive manufacturing of three-dimensional structures can be improved by removing excess sheath fluid from the receiving surface and, optionally, from the surface of the dispensed fiber structure, by a fluid removal component.

[0128] Excess fluid can be removed from the receiving surface or from the surfaces of the one or more layers of dispensed fibers by drawing the fluid away from those surfaces, by allowing or promoting evaporation of the fluid from those surfaces, or, in embodiments where the receiving surface is porous, by drawing the excess fluid through the porous surface. In some embodiments, the receiving surface comprises a porous material having pores sized to facilitate the passage of fluid therethrough and sized to support the one or more layers of fibrous structure deposited thereon.

[0129] In some embodiments, a component for removing excess fluid from a receiving surface and, optionally, from the surface of a distributed fiber structure can be included in a system configured to distribute material into a multi-walled plate or culture dish. In some embodiments, the receiving surface on the print bed includes an absorbent material or is placed near an absorbent material, which helps absorb excess fluid from the receiving surface. For example, a well plate insert having a bottom made of a porous membrane material or any other porous membrane substrate can be placed on top of or near an absorbent material such as a sponge. The absorbent material is used to absorb excess fluid from the receiving surface. In embodiments where the absorbent material is disposed below the porous receiving surface, excess fluid on the receiving surface can be drawn into the absorbent material through the porous receiving surface, thereby preventing excess fluid from pooling on the receiving surface. In embodiments where the absorbent material is disposed directly next to or on top of a portion of the receiving surface (e.g., at the periphery of the receiving surface so as not to interfere with the deposition of the distributed material), excess sheath fluid can be drawn from the receiving surface into the absorbent material.

[0130] In some embodiments, the receiving surface includes one or more tubes that are fluidically coupled to a vacuum source, and the vacuum source can provide suction to remove excess fluid from the receiving surface and optionally from the surface of the fiber structure of distribution. In such embodiments, a solid or porous receiving surface can also be used. In some embodiments, the print head is configured to further include one or more vacuum channels, each of which has an orifice located near (i.e., adjacent to) the distribution orifice. One or more vacuum channels each have an inlet, which is configured to facilitate communication with one or more vacuum fluids. When the print head is communicated with the vacuum fluid, one or more vacuum channels direct negative pressure to a portion of the surface area of ​​the fiber structure of the receiving surface being distributed or having been distributed from the distribution orifice, thereby extracting excess fluid from the surface of the fiber structure of distribution, thereby eliminating the convergence of fluid on the receiving surface and / or the fiber structure of distribution.

[0131] In some embodiments, the one or more vacuum tubes are at least partially disposed in one or more extensions protruding from the print head that protrude in the same general direction as the extension that includes the dispensing orifice and dispensing channel 110. In such embodiments, the one or more extensions that include the vacuum tubes do not extend further than the extension that includes the dispensing orifice and dispensing channel 110 so as not to interfere with the dispensing process.

[0132] In some embodiments, the fluid removal characteristic can be a characteristic of the fluid composition itself. For example, the sheath fluid composition and / or the buffer solution composition can be designed to evaporate after it is dispensed from the dispensing orifice, thereby eliminating the pooling of excess fluid on the receiving surface or the surface of the dispensed fibrous structure. For example, the sheath fluid can have a boiling point that causes it to evaporate after dispensing, while remaining in a liquid state prior to dispensing.

[0133] In some embodiments, the printing system includes a 3D electric stage comprising three arms for positioning a print head and a dispensing orifice in three-dimensional space above a print bed comprising a surface for receiving printed material. In one embodiment, the 3D electric stage (i.e., a positioning unit) can be controlled to position a vertical arm extending along the z-axis of the 3D electric stage so that the print head orifice points downward. A first horizontal arm extending along the x-axis of the electric stage is fixed to a fixed base platform. A second horizontal arm extending along the y-axis of the electric stage is movably coupled to the upper surface of the first horizontal arm so that the longitudinal directions of the first and second horizontal arms are perpendicular to each other. It should be understood that the terms "vertical" and "horizontal" used above with respect to the arms are intended to describe the manner in which the print head moves, and do not necessarily limit the physical orientation of the arms themselves.

[0134] In some embodiments, the receiving surface is located on top of a platform that is coupled to the upper surface of a second horizontal arm. In some embodiments, the 3D motorized stage arms are each driven by three corresponding motors and controlled by, for example, a programmable control processor of a computer. In a preferred embodiment, the print head and receiving surface are moved jointly along all three principal axes of a Cartesian coordinate system by a 3D motorized stage, and the movement of the stage is defined using computer software. It should be understood that the present invention is not limited to the positioning system described, and other positioning systems are known in the art. When material is dispensed from the dispensing orifice on the print head, the positioning unit moves in a pattern controlled by the software, thereby producing a first layer of the dispensed material on the receiving surface. Additional layers of the dispensed material are then stacked on top of one another, so that the final 3D geometry of the dispensed material layers is generally a replica of the 3D geometric design provided by the software. The 3D design can be created using typical 3D CAD (computer-aided design) software or generated from a digital image, as is known in the art. Furthermore, if the software-generated geometry contains information about the specific material to be used, then according to one embodiment of the present invention, specific input material types can be assigned to different geometric positions. For example, in some embodiments, a printed 3D structure can include two or more different input materials, where each input material has different properties (e.g., each input material includes a different cell type, a different cell concentration, a different ECM composition, etc.).

[0135] Aspects of the printing system of the present invention include software programs that are configured to facilitate the deposition of the input material of the present invention in a specific pattern and at a specific location to form a specific fiber, plane or 3D structure. In order to manufacture such structures, the printing system of the present invention deposits the input material of the present invention at a precise position (in two or three dimensions) on the receiving surface. In some embodiments, the position at which the printing system deposits the material is defined by user input and translated into computer code. In some embodiments, the computer code includes an instruction sequence that can be executed in a central processing unit (CPU) of a digital processing device and is written to perform a specified task. In some embodiments, printing parameters including but not limited to printing fiber size, pump speed, movement speed of the print head positioning system, and crosslinking agent strength or concentration are defined by user input and translated into computer code. In some embodiments, the printing parameters are not directly defined by user input, but are derived from other parameters and conditions by computer code.

[0136] Aspects of the present invention include methods for manufacturing tissue structures, tissues and organs, comprising: a computer module receiving input of a visual representation of a desired tissue structure; the computer module generating a series of commands, wherein the commands are based on the visual representation and can be read by a printing system of the present invention; the computer module providing the series of commands to the printing system; and the printing system depositing one or more input materials in accordance with the commands to form a structure having a defined geometry.

[0137] In some embodiments, the position where the printing system deposits input material is defined by user input and is translated into computer code. In some embodiments, the devices, systems, and methods disclosed herein also include a non-transitory computer-readable storage medium or a storage medium encoded with a 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 its components) or a computer connected to a bioprinter (or its components). In some embodiments, the computer-readable storage medium is optionally removable from the digital processing device. In some embodiments, as non-limiting examples, the computer-readable storage medium includes a CD-ROM, a DVD, a flash memory device, a solid-state memory, a disk drive, a tape drive, an optical drive, a cloud computing system, and / or services, etc. In some cases, programs and instructions are permanently, substantially permanently, semi-permanently, or non-temporarily encoded on a storage medium.

[0138] 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 a code segment) that interacts with a larger computer system. In some embodiments, a software module (or program module) appears in the form of one or more files and generally handles a specific task within a larger software system.

[0139] In some embodiments, the module is included in one or more software systems. In some embodiments, the module is integrated into one or more software systems with one or more other modules. A computer module is optionally an independent code segment, or optionally a code that cannot be individually identified. In some embodiments, the module is in a single application. In other embodiments, the module is in multiple applications. In some embodiments, the module is hosted on a single machine. In some embodiments, the module is hosted on multiple machines. In some embodiments, the module is hosted on multiple machines in one location. In some embodiments, the module is hosted on multiple machines in more than one location. A computer module according to an embodiment of the present invention allows an end user to use a computer to perform one or more aspects of the method described herein.

[0140] In some embodiments, the computer module includes a graphical user interface (GUI). As used herein, a "graphical user interface" refers to a user environment that uses images and text representations of the input and output of an application program and a hierarchical or other data structure of stored information. In some embodiments, the computer module includes a display screen. In other embodiments, the computer module presents a two-dimensional GUI through a display screen. In some embodiments, the computer module presents a three-dimensional GUI, such as a virtual reality environment, through a display screen. In some embodiments, the display screen is a touch screen and presents an interactive GUI.

[0141] Aspects of the present invention include one or more quality control components configured to monitor and / or adjust one or more parameters of the printing system of the present invention to ensure that 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 or outside the distribution channel 110 after distribution has occurred. In some embodiments, the quality control component includes 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., reducing pressure and / or reducing deposition speed) to reduce or avoid the formation of coiled structures by the printed fiber structure.

[0142] Aspects of the present invention include one or more fluid reservoirs, which are configured to store fluid and transport fluid to a printing system (e.g., printhead) via one or more fluid channels, which provide for fluid communication between the printing system and the reservoir. In some embodiments, the printing system includes one or more fluid reservoirs that are in fluid communication with the fluid channel. In some embodiments, the fluid reservoir is connected to an input orifice of the fluid channel. In some embodiments, the fluid reservoir is configured to accommodate a fluid volume ranging from about 100 μL to about 1L, such as 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 such as about 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or 950 mL.

[0143] In some embodiments, the printing system includes a pressure control unit that is fluidically coupled to one or more reservoirs. The pressure control unit is configured to provide force to move one or more fluids through the printing system. In some embodiments, the pressure control unit provides air pressure to the one or more fluids through one or more connecting tubes. The applied pressure forces the fluid to flow out of the reservoir and into the printhead through corresponding fluid channels. In some embodiments, alternative components can be used to move the fluid through the channel. For example, a series of electronically controlled syringe pumps can be used to provide the force to move the fluid through the printhead.

[0144] In some embodiments, the printing system includes a light module (as described above) for optionally exposing the photocrosslinkable input material to light to crosslink the material.Light modules according to embodiments of the present invention may be integrated into a printhead or may be a component of a printing system.

[0145] Input Materials:

[0146] Aspects of the present invention include input materials that can be used to print fiber structures. In some embodiments, the input material includes a hydrogel. Non-limiting examples of hydrogels include alginate, agarose, collagen, fibrinogen, gelatin, chitosan, hyaluronic acid-based gels, or any combination thereof. A variety of synthetic hydrogels are known and can be used in the 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 cell types, which can be dispersed within the hydrogel or added to the hydrogel after it is printed in a three-dimensional structure. In some embodiments, the hydrogel can be cross-linked by a chemical cross-linking agent. For example, a hydrogel including alginate can be cross-linked in the presence of divalent cations, a hydrogel including chitosan can be cross-linked using multivalent anions such as sodium tripolyphosphate (STP), a hydrogel including fibrinogen can be cross-linked in the presence of enzymes such as thrombin, and a hydrogel including collagen, gelatin, agarose or chitosan can be cross-linked in the presence of heat or an alkaline solution. In some embodiments, hydrogel fibers can be produced by a precipitation reaction by solvent extraction from an input material upon exposure to a crosslinker material that is miscible with 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 can form precipitation-mediated hydrogel fibers include polyethylene glycol (PEG) and alginate. Crosslinking of the hydrogel increases the hardness of the hydrogel, thereby allowing the formation of a solidified hydrogel in some embodiments.

[0147] In some embodiments, the hydrogel comprises alginate. When contacted with divalent cations, the alginate forms a solidified colloidal gel (high water content gel or hydrogel). Any suitable divalent cation can be used to form a solidified hydrogel with an input material comprising alginate. 2+ >Ba 2+ >Cu 2+ >Ca 2+ >Ni 2+ >Co 2+ >Mn 2+ In, Ca 2+ The most characteristic and most commonly used to form alginate gels (Ouwerx, C et al., Polymer Gels and Networks 1998, 6(5):393-408). Studies have shown that calcium alginate gels synergistically bind Ca through poly G blocks on adjacent polymer chains. 2+ ion formation, the so-called "egg box" model (ISP Alginates, Section 3: Manufacturing and Structure of Alginates, Alginates: Scientific Control Products, 2000, International Specialty Products: San Diego, pp. 4-7). G-rich alginates tend to form heat-stable, strong and brittle calcium gels, while M-rich alginates tend to form heat-stable, weaker but more elastic gels. In some embodiments, the hydrogel comprises a depolymerized alginate as described in U.S. Provisional Patent Application No. 62 / 437,601, the disclosure of which is incorporated herein by reference in its entirety.

[0148] In some embodiments, the hydrogel can be cross-linked using free radical polymerization to create covalent bonds between molecules. Free radicals can be generated by exposing a photoinitiator to light (usually ultraviolet light), or by exposing a hydrogel precursor to a chemical source of free radicals, such as ammonium peroxydisulfate (APS) or potassium peroxydisulfate (KPS) in combination with N,N,N,N-tetramethylethylenediamine (TEMED) as an initiator and catalyst, respectively. Non-limiting examples of photocrosslinkable hydrogels include methacrylated hydrogels, such as gelatin methacrylate (GEL-MA) or hydrogels based on polyethylene glycol diacrylate (PEG-DA), which are used in cell biology due to their ability to crosslink in the presence of free radicals after exposure to ultraviolet light and due to their inertness to cells. PEG-DA is often used as a scaffold in tissue engineering because polymerization occurs rapidly at room temperature and requires low energy input, has a high water content, is elastic, and can be customized to contain a variety of biomolecules.

[0149] Additional components:

[0150] The input material according to an embodiment of the present invention may include any of a variety of natural or synthetic polymers that support the vitality of living cells, including, for example, laminin, fibrin, hyaluronic acid, polyethylene glycol-based gels, gelatin, chitosan, agarose, or a combination thereof. In a particularly preferred embodiment, the bio-ink composition of the present invention is physiologically compatible, that is, it helps cell growth, differentiation, and communication. In certain embodiments, the input material includes one or more physiological matrix materials or a combination thereof. "Physiological matrix material" refers to a biological material 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), adhesion glycoproteins, and collagens (e.g., collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, or collagen XVIII).

[0151] Collagen provides tensile strength for most tissues, and multiple collagen fibrils with a diameter of about 100 nm combine to form strong coiled coil fibers with a diameter of about 10 μm. The biomechanical functions of certain tissue structures are imparted by the oriented arrangement of collagen fibers. In some embodiments, the input material includes collagen fibrils. The input material including collagen fibrils can be used to generate the fiber structure that forms the tissue structure. By adjusting the diameter of the fiber structure, the orientation of the collagen fibrils can be controlled, thereby guiding the polymerization of the collagen fibrils in a desired manner.

[0152] For example, previous studies have shown that microfluidic channels of varying diameters can direct the polymerization of collagen fibrils to form fibers oriented 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 along the direction of the collagen fibers. In another study, Martinez et al. demonstrated that 500 μm channels within beaded cellulose scaffolds could direct the alignment of collagen and cells (Martinez et al., 2012). In some embodiments, the input material can be formed into a fiber structure having a diameter ranging from about 20 μm to about 500 μm, such as 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 the collagen fibers within the fiber structure can be controlled. Thus, the fiber structure and the collagen fibers within the fiber structure can be patterned to produce a tissue construct with a desired collagen fiber arrangement, which is critical for imparting desired biomechanical properties to the 3D printed structure.

[0153] Mammalian cell types:

[0154] Input material according to embodiments of the present invention can be combined with any mammalian cell type, including but not limited to stem cells (e.g., embryonic stem cells, adult stem cells, induced pluripotent stem cells), germ cells, endoderm cells (e.g., lung, liver, pancreas, gastrointestinal or urogenital tract cells), mesodermal cells (e.g., kidney, bone, muscle, endothelial or cardiac cells) and ectoderm cells (skin, nervous system or eye cells), or any combination thereof.

[0155] 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, cardiomyocytes, epithelial cells, endothelial cells, myoblasts, chondroblasts, osteoblasts, osteoclasts, and any combination thereof.

[0156] Cells can be obtained from donors (exogenous) or recipients (autologous). Cells can also be from established cell culture lines, or can be cells that have been genetically engineered and / or manipulated to achieve a desired genotype or phenotype. In some embodiments, tissue sheets can also be used that can provide a variety of different cell types within the same structure.

[0157] In some embodiments, the cells can be obtained from a suitable donor (human or animal) or from a subject into which the cells will be implanted. 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 other embodiments, the cells can be derived from animals, such as dogs, cats, horses, monkeys, or any other mammals.

[0158] Suitable growth conditions for mammalian cells are well known in the art (Freshney, RI (2000) Animal Cell Culture, A Handbook of Basic Techniques. John Wiley & Sons, Hoboken, NJ; Lanza et al., Principles of Tissue Engineering, Academic Press; 2nd ed. May 15, 2000; and Lanza & Atala, Tissue Engineering Methods, Academic Press; 1st ed. October 2001). Cell culture media typically include essential nutrients and optional additional elements, such as growth factors, salts, minerals, vitamins, etc., which can be selected based on the cell type being cultured. Specific components can be selected to enhance cell growth, differentiation, secretion of specific proteins, etc. Typically, standard growth media include Dulbecco's Modified Eagle's Medium, low glucose (DMEM), containing 110 mg / L pyruvate and glutamine, supplemented with 10% to 20% fetal bovine serum (FBS) or calf serum and 100 U / ml penicillin, as well as various other standard culture media known to those skilled in the art. Growth conditions will vary depending on the mammalian cell type used and the desired tissue.

[0159] In some embodiments, cell type specific reagents can be advantageously used in the subject input material for the corresponding cell type. For example, the extracellular matrix ("ECM") can be extracted directly from the tissue of interest and then dissolved and incorporated into the input material to generate a tissue specific input material for printing tissue. Such ECM can be readily obtained from patient samples and / or can be obtained from, for example, zPredicta (rBone TM , available at zpredicta.com / home / products).

[0160] Active agent:

[0161] In some aspects, the input material according to embodiments of the present invention may comprise at least one active agent. Non-limiting examples of such active agents 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, pleiotropic growth factor, endothelin, nicotinamide, glucagon-like peptide-I, glucagon-like peptide-II, parathyroid hormone, tenascin-C, tropoelastin, thrombin-derived peptide, laminin, biological peptides containing cell binding domains and biological peptides containing heparin binding domains, therapeutic agents, and any combination thereof.

[0162] As used herein, the term "therapeutic agent" refers to any chemical moiety that is a biological, physiological 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 references such as The Merck Index, The Physician's Desk Manual, and The Pharmacological Basis of Therapeutics, and they include, but are not limited to, drugs; vitamins; mineral supplements; substances used to treat, prevent, diagnose, cure, or alleviate a disease or illness; substances that affect the structure or function of the body; or prodrugs that become biologically active or more active after being placed in a physiological environment. In some embodiments, one or more therapeutic agents can be used that are capable of being released from an input material described herein into adjacent tissues or fluids after being implanted 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.

[0163] The additional active agent can include, but is not limited to, a protein, a peptide, a nucleic acid analog, a nucleotide, an oligonucleotide, a nucleic acid (DNA, RNA, siRNA), a peptide nucleic acid, an aptamer, an antibody or fragment or portion thereof, an antigen or epitope, a hormone, a hormone antagonist, a growth factor or recombinant growth factor and fragments and variants thereof, a cytokine, an enzyme, an antibiotic or antimicrobial compound, an anti-inflammatory agent, an antifungal agent, an antiviral agent, a toxin, a prodrug, a small molecule, a drug (e.g., a drug, a dye, an amino acid, a vitamin, an antioxidant), or any combination thereof.

[0164] 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, cefditoren, ceftobiprole), glycopeptides (e.g., vancomycin), macrolides (e.g., erythromycin, azithromycin), monolactams, penicillins (e.g., amoxicillin, ampicillin, cloxacillin, dicloxacillin, flucloxacillin), polypeptides (e.g., bacitracin, polymyxin B), quinolones (e.g., quinolones ... sulfonamides (e.g., sulfasalazine, trimethoprim, trimethoprim-sulfamethoxazole (cotrimoxazole)), tetracyclines (e.g., doxycycline, minocycline, tetracycline, etc.), chloramphenicol, lincomycin, clindamycin, ethambutol, mupirocin, metronidazole, pyrazinamide, thiamphenicol, rifampicin, thiamphenicol, dapsone, clofazimine, quinupristin, metronidazole, linezolid, isoniazid, fosfomycin, fusidic acid, or any combination thereof.

[0165] Non-limiting examples of antibodies include: abciximab, adalimumab, alemtuzumab, basiliximab, bevacizumab, cetuximab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, ibritumomab tiuxetan, infliximab, muromona-CD3, natalizumab, ofatumumab, omalizumab, palivizumab, panitumumab, ranibizumab, rituximab, tositumomab, trastuzumab, pentetate, atumab, acitretin, tocilizumab, belimumab, bevacizumab, and bisoprolol.

[00155] The present invention relates to an agent selected from the group consisting of: abacterial, levofloxacin, canakinumab, caprotuzumab pendetide, catumaxomab, denosumab, edrecolomab, ifenguzumab, ertuinomab, daclizumab, fasotuzumab, fontuzumab, gemtuzumab, insinuomab, labetuzumab, mepolizumab, motuzumab, nimotuzumab, nofetumomab, ogovuzumab, pertuzumab, rovizumab, lulizumab, thiosomab, temetuzumab, tefetumomab, tocilizumab, ustekinumab, visilizumab, futumomab, zalumab, zalumab, or any combination thereof.

[0166] Non-limiting examples of enzymes suitable for use with the input materials described herein include peroxidases, lipases, amylose, organophosphate dehydrogenases, ligases, restriction endonucleases, ribonucleases, DNA polymerases, glucose oxidase, and laccase.

[0167] Additional non-limiting examples of active agents suitable for use with the input materials of the present invention include: cell growth media, such as Dulbecco's modified Eagle's medium, fetal bovine serum, non-essential amino acids and antibiotics; growth 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 morphogenic growth factor, bone morphogenetic protein, transforming growth factor, nerve growth factor and related proteins (growth factors are known in the art, see, for example, Rosen & Thies, Cellular and Molecular Basis of Bone Formation and Repair (RG Landes Co., Austin, Texas, 1995); anti-angiogenic proteins, such as endostatin and other naturally derived or genetically engineered proteins; polysaccharides, glycoproteins or lipoproteins; anti-infective agents, such as antibiotics and antiviral agents, chemotherapeutic agents (i.e., anticancer agents), anti-rejection agents, analgesics and analgesic combinations, anti-inflammatory agents, steroids, or any combination thereof.

[0168] Additional fluid:

[0169] Aspects of the present invention include one or more buffer solutions. Buffer solutions according to embodiments of the present invention are miscible with the input material (e.g., a hydrogel) and do not crosslink the input material. In some embodiments, the buffer solution comprises an aqueous solvent. Non-limiting examples of buffer solutions include polyvinyl alcohol, water, glycerol, propylene glycol, sucrose, gelatin, or any combination thereof.

[0170] 500, 3,750, 4,000, 4,250, 4,500, or 4,750 mPa s. In some embodiments, the viscosity of the buffer solution can be adjusted to match the viscosity of one or more input materials.

[0171] 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, at least in part, to encapsulate or "coat" the 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, glycerin, propylene glycol, sucrose, gelatin, or any combination thereof. The viscosity of the sheath fluid according to embodiments of the present invention can range from about 1 mPa·s to about 5,000 mPa·s, for example, 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.

[0172] In some embodiments, the sheath fluid includes a chemical crosslinking agent. In some embodiments, the chemical crosslinking agent includes a divalent cation. Non-limiting examples of divalent cations include Cd 2+ 、Ba 2+ 、Cu 2+ , Ca 2+ 、Ni 2+ 、Co 2+ or Mn 2+ In a preferred embodiment, Ca 2+ As divalent cations. In some embodiments, the concentration of divalent cations in the sheath fluid ranges from about 80 mM to about 140 mM, such as about 90, 100, 110, 120, or 130 mM.

[0173] Directions:

[0174] Aspects of the present invention include methods for printing a linear fiber structure, a planar structure including one or more fiber structures, or a three-dimensional (3D) structure including two or more layers of planar structure. In some embodiments, a method first includes providing a design for the plane or 3D structure to be printed. The design can be created using commercially available CAD software. In some embodiments, the design includes information about specific materials (e.g., for heterogeneous structures including multiple materials), which will be assigned to specific locations in the structure to be printed.

[0175] In some embodiments, a method includes using a 3D printer, the printer comprising: a print head; a receiving surface for receiving material dispensed by the print head; and a positioning unit operably coupled to the receiving surface, the positioning unit for positioning the print head at a position in three-dimensional space above the receiving surface. For example, various embodiments of the printing system provided herein can be used in methods for printing planar or 3D structures.

[0176] Aspects of the method include providing one or more input materials to be dispensed by a printhead. In some embodiments, the one or more cell types are compatible with the input materials and are optionally dispensed within the input materials. In some embodiments, a sheath fluid acts as a lubricant to lubricate movement of the input material within the printhead. In some embodiments, the sheath fluid includes a crosslinker to solidify at least a portion of the hydrogel prior to or during dispensing from the printhead.

[0177] Aspects of the method include transmitting a design to a 3D printer. In some embodiments, communication can be achieved, for example, via a programmable control processor. In some embodiments, the method includes controlling the relative positioning of a print head and a receiving surface in three-dimensional space, and simultaneously dispensing sheath fluid and input material from the print head, either individually or in combination. In some embodiments, the material dispensed from the print head is coaxially dispensed such that the sheath fluid encapsulates the input material. This coaxial arrangement allows a crosslinker in the sheath fluid to cure the input material, thereby producing a cured fiber structure dispensed from the print head.

[0178] In some embodiments, a method includes: depositing a first layer of a distributed fiber structure on a receiving surface, the first layer including an arrangement of fiber structures specified by a design, and iteratively repeating the depositing step; depositing subsequent fiber structures onto the first and subsequent layers, thereby depositing the distributed fiber structure layer by layer in a geometric arrangement specified by the design to produce a 3D structure.

[0179] In some embodiments, multiple input materials, such as multiple hydrogels, at least some of which include one or more cell types, are deposited in a controlled sequence, thereby allowing controlled deposition of input materials and cell types in a design-specified geometric arrangement.

[0180] In some embodiments, a method comprises removing excess fluid from a receiving surface and optionally from the surface of the fiber structure of distribution. For example, the step of removing excess fluid can be continuously carried out in the whole printing process, thereby removing excess fluid, otherwise these excess fluids may interfere with the fiber structure of distribution being layered in the geometric arrangement provided by the design. Alternatively, the step of removing excess fluid can be carried out intermittently or simultaneously in sequence with one or more deposition steps in the whole printing process. In some embodiments, by extracting fluid from a receiving surface and optionally from the surface of the fiber structure of distribution and removing excess fluid. In some embodiments, by removing excess fluid by being sucked through a receiving surface, the receiving surface comprises a hole that is dimensioned to allow fluid to pass through. In some embodiments, removing excess fluid is realized by providing the fluid that evaporates after being distributed from the distribution orifice.

[0181] Aspects of the present invention include methods of making 3D structures comprising one or more input materials.The 3D structures can be used to repair and / or replace at least a portion of damaged or diseased tissue in a subject.

[0182] As described above, any suitable divalent cation may be used in conjunction with the methods of the present invention to cure chemically cross-linkable input materials, including but not limited to Cd 2+ 、Ba 2+ 、Cu 2+ , Ca 2+ 、Ni 2+ 、Co 2+ or Mn 2+ In a preferred embodiment, Ca 2+ In a preferred embodiment, the chemically cross-linkable input material is combined with a Ca 2+ In some embodiments, the Ca in the sheath fluid is contacted with the solidified fiber structure. 2+ The concentration of ranges from about 80 mM to about 140 mM, such as about 90, 100, 110, 120, or 130 mM.

[0183] In certain embodiments, the input material solidifies in less than about 5 seconds, eg, less than about 4 seconds, less than about 3 seconds, less than about 2 seconds, or less than about 1 second.

[0184] Aspects of the present invention include methods for depositing one or more input materials in a patterned manner using software tools to form a solidified structural layer, which is formed into a multilayer 3D tissue structure. In some embodiments, the multilayer 3D tissue structure comprises a plurality of mammalian cells. Advantageously, by adjusting the components of the input materials of the present invention (e.g., mammalian cell type, cell density, matrix components, active agents), the methods of the present invention can be used to create a multilayer 3D tissue structure, wherein the multilayer 3D tissue structure has a precisely controlled composition at any specific location in three-dimensional space. Therefore, the methods of the present invention facilitate the production of complex three-dimensional tissue structures.

[0185] In some embodiments, the method includes simultaneously dispensing a buffer solution and / or sheath fluid through the core channel, one or more input materials through one or more shell channels, and sheath fluid through the sheath flow channel 118 to form a hollow core in the printed fiber.

[0186] 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 to solidify the outer surface of the crosslinkable material flow in the distribution channel 110.

[0187] In some embodiments, the non-crosslinkable material in the core channel comprises a chemical crosslinker and the sheath fluid in the sheath flow channel 118 comprises an aqueous solvent, and contact occurs at the first fluid focusing intersection to solidify the inner surface of the crosslinkable material flow in the distribution channel 110.

[0188] In some embodiments, the non-crosslinkable material in the core channel includes a chemical crosslinker and the sheath fluid in the sheath flow channel 118 includes a chemical crosslinker, and contact occurs at the first fluid focusing intersection to solidify the inner surface of the crosslinkable material flow, and contact occurs at the sheath fluid intersection to solidify the outer surface of the crosslinkable material flow in the distribution channel 110.

[0189] In some embodiments, the system includes a print head comprising a core channel comprising at least two core inlet sub-channels connected to different fluid reservoirs comprising different input materials, and the method includes alternately dispensing the different input materials through the shell inlet sub-channels 126 to produce a cured fiber structure comprising different core materials along the length of the continuous fiber.

[0190] In some embodiments, the system includes a print head comprising a first shell channel, the first shell channel comprising at least two shell inlet sub-channels 126, the shell inlet sub-channels connected to different fluid reservoirs comprising different materials, and the method includes alternately dispensing different input materials through the shell inlet sub-channels 126 to produce a cured fiber structure comprising different shell materials along the length of the continuous fiber.

[0191] In some embodiments, the system includes a printhead comprising at least two shell channels connected to different fluid reservoirs comprising different input materials, and the method includes simultaneously dispensing the different input materials through the first and second shell channels to produce a cured fiber structure comprising different concentric shells.

[0192] In some embodiments, the system includes a printhead including a first shell channel including at least two shell inlet subchannels 126 connected to different fluid reservoirs including a reinforcing hydrogel material and a biocompatible hydrogel material, and the method includes alternately dispensing the reinforcing hydrogel material and the biocompatible hydrogel material through the dispensing channel 110 to produce perfusable tissue fibers. In another embodiment, the printhead also includes a second shell channel 128, and the method also includes dispensing the same or different reinforcing hydrogel material through the second shell channel 128 to produce a concentric second shell surrounding the first shell.

[0193] In an alternative embodiment, solid-core fibers can be produced as a means of printing otherwise unprintable materials. This approach allows for the selection of easily printable shell materials, such as alginate, and the selection of otherwise unprintable core materials, such as pure collagen. The ability to switch core materials allows users to further control core composition. The core material can contain different cell types and be sequenced or combined along the fiber length. In this case, the shell material can also be switched and can also contain different cell types.

[0194] Practicality:

[0195] In some embodiments, the structure using the system and method that this paper provides to produce can be used for the field of drug discovery, wherein for example determining that the reaction of cell to various compounds and compositions is significant.The plane that uses the embodiment of the system and method that this paper provides to make and the use of 3D cell culture can provide the experimental condition that is more similar to cell and tissue condition in vivo with respect to traditional 2D cell culture.The 3D arrangement of cell can more closely simulate cell-cell interaction and the reaction to external stimulus in vivo, and can use the heterogeneity of the 3D structure that the system and method that this paper provides to produce to allow research tissue and potential organ.The structure that carries 3D cell that expection uses the embodiment of the system and method that this paper provides to make can provide similar benefit for cosmetics industry by providing the alternative means of test cosmetics.

[0196] In some embodiments, each aspect of the system and method provided herein is compatible with standard orifice plate technology. In the method and system provided herein, the orifice plate or orifice plate insert can be used together with a print bed or as a part of a print bed. Therefore, the various embodiments of the system and method provided herein are compatible with instruments and practices utilizing orifice plates, thereby allowing them to be easily integrated into existing process streams.

[0197] In some embodiments, one or more fluid channels within the printhead of the present invention are compatible with other microfluidic modules. For example, a known microfluidic module can be included in the printhead of the system provided herein upstream of the dispensing orifice. Such modules can include, for example, cell counting, cell sorting, cell analysis, and / or concentration gradient generation modules.

[0198] In some embodiments, the throughput of 3D printing can be increased by adding additional print heads to the system in parallel. Each print head includes all the elements required to print a multi-material structure, so multiple 3D structures can be printed simultaneously by including additional print heads in the system.

[0199] All patents and patent applications cited herein are hereby incorporated by reference in their entirety.

[0200] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one skilled in the art will readily appreciate in light of the teachings of this invention that certain changes and modifications may be made without departing from the spirit or scope of the appended claims.

[0201] The present invention realizes multi-material switching, therefore can modify the composition (cell type and biomaterial composition) of vascular wall along the length of passage when printing continuously.This example is for reproducing the biological structure and function of renal tubule, and the wall composition at proximal end will be different from the wall composition at distal end.Or in perfusable printing 3D liver tissue, wherein vascular wall may be lined with low permeability portal vein endothelial cells at the larger open end of blood vessel, and wherein in the narrower blood vessel of passage, there is the sinusoidal endothelial cell of higher permeability to model sinusoidal space.Similar to liver tissue, it is necessary to study the interaction of the different stromal cell types of the passage outside in the content of perfusion channel and one or more shells.This can be applied to produce the multi-tissue toxicity model combining shear flow effect.Single tissue can be printed, wherein the cell content of fiber shell switches along its length, to produce the coded hollow fiber with the different regions corresponding to different organ types.The switching of this shell content is impossible for the system based on non-microfluidic syringe.

[0202] Example:

[0203] Example 1: Perfusable Tissue Fibers

[0204] Bioprinted perfusable tissue fibers with a liquid core and a cell-containing gel shell have significant commercial and clinical significance, making it possible to insert a needle into the core of the fiber and attach to a pump after printing that pushes the fluid of interest through the fiber. In this way, one can simulate the flow of nutrients, drugs, or other compounds of interest through the cell-containing fiber. Unfortunately, however, a major challenge arises when connecting the needle to the fiber because the mechanical requirements for making this connection are very different from those required to support functional biology. Therefore, the ability to switch shell materials in real time during the printing process allows the user to print with a strong material in the area where the needle is intended to be connected (the fiber end), and print with a soft cell-containing material for the area that is intended to support biological function.

[0205] like Figure 12 As shown in Figure 1, perfusable tissue fibers according to the present invention were bioprinted using a reinforcing hydrogel material (blue) composed of 4 wt% low-viscosity sodium alginate, a biocompatible hydrogel material (red) composed 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 to the fiber, and a mixture of gelatin and transglutaminase was used to seal the needle / fiber connection to prevent leakage during perfusion.

[0206] Furthermore, even with adequate reinforcement at the ends of the perfusable fibers to achieve proper connections, breakage can occur with increasing length of fibers comprising softer cell-laden materials, particularly at higher flow rates. Figure 13As shown in , the present invention further contemplates the optional addition of a second concentric shell comprising the same or different reinforcing hydrogel material such that the fiber is supported along its entire length.

[0207] The foregoing merely illustrates the principles of the present invention. It should be understood that those skilled in the art will be able to design various arrangements that, although not explicitly described or shown herein, embody the principles of the present invention and are included within its spirit and scope. In addition, all embodiments and conditional language described herein are primarily intended to help the reader understand the principles of the present invention and the conception of the promotion technology provided by the inventor, and should be interpreted as not limiting the embodiments and conditions of such specific descriptions. In addition, all statements describing the principles and aspects of the present invention and its specific embodiments herein are intended to encompass both their structural equivalents and functional equivalents. In addition, it is intended that such equivalents include currently known equivalents and equivalents developed in the future, i.e., any element that performs the same function, regardless of the structure. Therefore, the scope of the present invention is not intended to be limited to the exemplary aspects shown and described herein. On the contrary, the scope and spirit of the present invention are embodied by the appended claims.

Claims

1. A print head for a three-dimensional printer, the print head comprising a plurality of stacked layers forming a plurality of fluid channels, the print head comprising: a core channel having at least one inlet and an outlet; a first shell passage, the first shell passage having at least one inlet and an outlet; Multi-channel housing; as well as Assign channels, wherein the core channel and / or the first shell channel further comprises at least one fluid distribution orifice configured to distribute fluid around a circumference of the core channel outlet and / or the first shell channel outlet, wherein the multi-channel housing comprises the core channel outlet, the first shell channel outlet and a first fluid focusing chamber, wherein the core channel outlet is disposed in a central region of the multi-channel housing and is in fluid communication with an inlet of the first fluid focusing chamber, wherein the core channel outlet extends to a first vertical depth into the multi-channel housing in alignment with the distribution channel, wherein the first shell channel outlet is concentrically disposed about the core channel and is in fluid communication with the inlet of the first fluid focusing chamber, wherein the first shell channel outlet extends to a second vertical depth in the multi-channel housing, and The first fluid focusing chamber converges toward the distribution channel and includes a frusto-conical shape configured to focus fluid toward the distribution channel. 2 . The print head of claim 1 , wherein the first housing channel outlet has a gradient width that increases with increasing depth into the multi-channel housing.

3. The printhead of claim 1 , wherein the first vertical depth is greater than the second vertical depth such that the core channel outlet extends further into the multi-channel housing and / or the first fluid focusing chamber than the first shell channel outlet.

4. The print head according to claim 1, further comprising: a second shell channel having at least one inlet, an outlet, and at least one fluid distribution orifice configured to distribute fluid around a circumference of the second shell channel outlet, wherein the second shell channel outlet is concentrically disposed about the first shell channel outlet in the multi-channel housing in a same layer of the printhead and is in fluid communication with the first fluid focusing chamber.

5. The printhead of claim 4, wherein the first shell channel outlet extends further into the multi-channel housing and / or the first fluid focusing chamber than the second shell channel outlet.

6. The print head according to claim 1, further comprising: a second shell channel having at least one inlet, an outlet, and at least one fluid distribution orifice configured to distribute fluid around a circumference of the second shell channel outlet, and a second multichannel housing positioned between the first fluid focusing chamber and a distal end of the distribution channel, wherein the second multi-channel housing comprises the distribution channel, the second shell channel outlet and the second fluid focusing chamber, wherein the distribution channel is disposed in a central region of the second multi-channel housing, is in fluid communication with an inlet of the second fluid focusing chamber, and extends to a first vertical depth into the multi-channel housing, wherein the second shell channel outlet is concentrically disposed about the distribution channel and is in fluid communication with the inlet of the second fluid focusing chamber and extends to a second vertical depth into the second multi-channel housing, and The second fluid focusing chamber converges toward the distribution channel and includes a frusto-conical shape configured to focus fluid toward the distribution channel.

7. The printhead of claim 6, wherein the second multi-channel housing overlaps the first fluid focusing chamber in the same layer of the printhead.

8. A print head according to claim 1, wherein the at least one fluid distribution orifice connects the core channel inlet and / or the first shell channel inlet with the vertex of the upper curved surface of the core channel outlet and / or the first shell channel outlet, wherein the upper curved surface has a parabolic or elliptical shape.

9. The print head according to claim 4, wherein the at least one fluid distribution orifice connects the first and / or second housing channel inlet with a vertex of an upper curved surface of the first and / or second housing channel outlet, wherein the upper curved surface has a parabolic or elliptical shape.

10. The print head of claim 6, wherein the at least one fluid dispensing orifice connects the second housing channel inlet with a vertex of an upper curved surface of the second housing channel outlet, wherein the upper curved surface has a parabolic or elliptical shape.

11. The printhead of claim 6, wherein the first and / or second housing channel inlets comprise two or more sub-channels configured to deliver fluid to the same fluid dispensing orifice or separate fluid dispensing orifices.

12. A print head according to claim 11, wherein each sub-channel comprises a fluid distribution orifice connecting the first and / or second shell channel inlet with the vertex of the upper curved surface of the corresponding first and / or second shell channel outlet, and wherein the upper curved surface has a parabolic or elliptical shape.

13. A printhead according to claim 11 or 12, wherein each subchannel is configured to dispense a different material.

14. The print head according to claim 6 further includes a sheath flow channel, which converges with the distribution channel at a sheath fluid chamber located between the first and second fluid focusing chambers and the distal end of the distribution channel; wherein the sheath fluid chamber includes a truncated conical shape, and the truncated conical shape is configured to focus the fluid toward the distribution channel. 15 . The print head according to claim 14 , wherein the sheath flow channel comprises a plurality of sheath flow sub-channels converging toward the distribution channel via the sheath fluid chamber.

16. The printhead of claim 1, wherein the printhead comprises at least two wick inlet subchannels that converge at or near the wick channel outlet, the multi-channel housing, and / or the fluid dispensing orifice.

17. A printhead according to claim 16, wherein the at least two wick inlet subchannels converge in an immediately preceding layer, or in the same layer of the printhead as the wick channel outlet, the multi-channel housing and / or the fluid distribution orifice.

18. A printhead according to claim 16 or 17, wherein each wick inlet sub-channel is configured to dispense a different material.

19. A printhead comprising a plurality of stacked layers forming a plurality of fluid channels, the printhead comprising: core channel; multiple shell channels; as well as a fluid focusing chamber converging toward the distribution channel, wherein the core channel is in fluid communication with the fluid focusing chamber, wherein the core channel extends longitudinally through a central region of the fluid focusing chamber and is aligned with the distribution channel, wherein the plurality of shell channels are concentrically disposed around the core channel in a same layer of the printhead and are in fluid communication with the fluid focusing chamber, wherein the inner shell channel extends longer into the fluid focusing chamber than the outer shell channel, wherein the core channel extends longer into the fluid focusing chamber than any shell channel, and The sheath flow channel where the sheath fluid chamber converges with the distribution channel is located between the fluid focusing chamber and the distal end of the distribution channel.

20. The print head according to claim 19, further comprising: A plurality of fluid distribution orifices are configured to distribute fluid around a circumference of the plurality of shell channels, wherein the plurality of fluid distribution orifices individually connect respective shell channel inlets with vertices of upper curved surfaces of corresponding shell channel outlets of the plurality of shell channels.

21. The printhead of claim 19, wherein at least one shell channel of the plurality of shell channels has a gradient width that increases with increasing longitudinal depth into the housing.

22. A printhead comprising a plurality of stacked layers forming a plurality of fluid channels, the printhead comprising: a core channel comprising at least two core inlet subchannels having distinct fluid reservoirs, input orifices, and control valves that converge to form a single core channel outlet in fluid communication with the first fluid focusing chamber; a first shell channel comprising at least two shell inlet subchannels having distinct fluid reservoirs, input orifices, and control valves that converge to form a single shell channel outlet in fluid communication with a second fluid focusing chamber; a distribution channel; wherein the first and second fluid focusing chambers converge toward the distribution channel, wherein the first and second fluid focusing chambers comprise a frustoconical shape configured to focus fluid toward the distribution channel; as well as a sheath flow channel that converges with the distribution channel at a sheath fluid intersection point between a second fluid focusing intersection point and the distal end of the distribution channel, The core channel and / or the first shell channel further comprises at least one fluid distribution orifice configured to distribute fluid around a circumference of the core channel outlet and / or the first shell channel outlet.

23. The printhead of claim 22, wherein the first shell channel comprises three shell inlet subchannels, one of which is connected to a fluid reservoir comprising a buffer solution.

24. A print head according to claim 22, wherein the at least one fluid distribution orifice connects the converging core inlet subchannel to the vertex of the upper curved surface of the core channel outlet, and / or connects the converging shell inlet subchannel to the vertex of the upper curved surface of the first shell channel outlet; wherein the upper curved surface has a parabolic or elliptical shape.

25. The printhead of claim 22, wherein the at least two wick inlet subchannels converge at or near the wick channel outlet.

26. A system for producing a fibrous structure, the system comprising: A print head according to any one of claims 1 to 25; as well as A positioning assembly is provided for positioning the dispensing orifice of the print head in three-dimensional space, wherein the positioning assembly is operatively coupled to the print head.

27. The system of claim 26, further comprising a programmable control processor for controlling the positioning assembly and for controlling the flow rate of one or more fluids through the print head.

28. The system of claim 26, further comprising a fluid removal assembly configured to remove excess fluid dispensed from the print head, wherein the fluid removal assembly comprises a porous membrane configured to allow the excess fluid to pass therethrough, and / or wherein the fluid removal assembly comprises a vacuum configured to draw the excess fluid.

29. The system of claim 26, further comprising a pressure control assembly configured to regulate a flow rate of one or more fluids through the printhead.

30. A method of producing a core-shell fiber structure, the method comprising: A system for producing a fiber structure is provided, the system comprising: The print head according to any one of claims 1 to 25, wherein the printhead is configured to dispense a plurality of input materials through the core channel and the sheath channel, wherein at least one of the input materials comprises a cross-linkable material, and a sheath solution through a sheath flow channel; a receiving surface for receiving a first layer of material dispensed from the print head; a positioning assembly for positioning a dispensing orifice of the print head in 3D space, wherein the positioning assembly is operatively coupled to the print head; a programmable control processor for controlling the positioning assembly and for controlling the flow rate of one or more fluids through the print head; a fluid reservoir comprising the plurality of input materials and a sheath solution, wherein the fluid reservoir is in fluid communication with the printhead; contacting the cross-linkable material with the sheath solution in the distribution channel to produce a solidified fiber structure; and The solidified fibrous structure is dispensed from the dispensing orifice of the print head.

31. A method according to claim 30, wherein the system includes a core channel, the core channel including at least two core inlet sub-channels, the core inlet sub-channels being connected to different fluid reservoirs including first and second input materials, respectively, and the method includes alternately dispensing the first and second input materials through the core inlet sub-channels to produce a solidified fiber structure including different core materials along the length of the continuous fiber.

32. A method according to claim 30 or 31, wherein the system includes a first shell channel, the first shell channel includes at least two shell inlet sub-channels, the shell inlet sub-channels are respectively connected to different fluid reservoirs including third and fourth input materials, and the method includes alternately dispensing the third and fourth input materials through the shell inlet sub-channels to produce a solidified fiber structure including different shell materials along the length of the continuous fiber.

33. A method according to claim 30 or 31, wherein the system comprises first and second shell channels, the shell channels being connected to different fluid reservoirs comprising third and fourth input materials, respectively, and the method comprises dispensing the third and fourth input materials through the first and second shell channels to produce a cured fiber structure comprising different concentric shells.

34. The method of claim 30, further comprising: encoding the programmable control processor with the planar structure to be printed; and A first layer of the solidified fiber structure is deposited on the receiving surface to print the planar structure.

35. The method of claim 34, further comprising: encoding the programmable control processor with the 3D structure to be printed; and Subsequent layers of the solidified fiber structure are deposited on top of the planar structure to print a 3D structure.

36. A bioprinted tissue fiber produced by the method of any one of claims 31 to 33, the bioprinted tissue fiber having variable core and sheath materials throughout the length of the fiber.

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