Systems and methods for printing core shell fibers

Abstract

Systems and methods for printing core shell fibers are provided. A printhead for a three-dimensional printer, in one embodiment, the printhead includes a multi-channel housing including a core channel outlet, a first housing channel outlet, and a first fluid focusing chamber converging toward a distribution channel, where the core channel outlet is in a central region of the housing, the first housing channel outlet is in a central region of the housing, and the first housing channel outlet is in a central region of the housing. And the core channel and the shell channel extend into the outer shell by respective depths. In another embodiment, the plurality of shell channels include an inner shell channel that extends longer than an outer shell channel into the focusing chamber, and a core channel that extends longer than any shell channel into the focusing chamber. In another embodiment, each of the core channel and the first shell channel includes at least two inlet sub-channels having different fluid reservoirs, input orifices, and control valves that converge to form a single outlet that communicates with a respective focusing chamber. A sheath flow channel may be provided.

Description

[0001] This application is a divisional application of the invention application filed on November 1, 2020, with Chinese national application number 202080091736.8, entitled "System and method for printing core shell fibers". Technical Field

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

[0003] Tissue engineering techniques have long sought to create viable synthetic structures capable of mimicking and / or replacing living organs and tissues using a multitude of materials and methods. The lack of pre-patterned vascular systems is one of the major factors limiting the success of current tissue engineering strategies, and the inability to fabricate thick tissue structures containing endogenous engineered vascular systems or nutrient channels that can integrate with host tissues is a major technical obstacle preventing the generation and / or implantation of larger, viable, and / or metabolically active tissues.

[0004] 3D printing, a form of additive manufacturing, has been applied to create three-dimensional objects directly from digital files, where the objects are built layer by layer to achieve the desired three-dimensional structure. Initial efforts to adapt these 3D printing techniques for creating patterned structures of hollow containers focused primarily on printing and subsequently removing sacrificial material. Bertassoni et al., for example, used a physical method to remove templated agarose from a surrounding mold of a photocrosslinked acrylic hydrogel (e.g., gelMA) (Lab on a Chip 14:2202 (2014)). The printed agarose fibers have very weak adhesion to the gelMA, but unfortunately, they must be removed manually, which is time-consuming, difficult, and requires the cast hydrogel to be stronger than the agarose fibers.

[0005] Alternative methods involve printing sacrificial fiber networks from materials, which can then be removed by solubilization or liquefaction. For example, Wu et al. printed 3D perfusionable vascular trees by extruding sacrificial Pluronic F127 filaments into Pluronic F127-diacrylate gel reservoirs to provide support during printing (Advanced Materials 2011; 23:H178-183). Unmodified Pluronic F127 channels, after photocuring the surrounding acrylate-modified Pluronic F127-diacrylate, can be liquefied by lowering the temperature below their critical micelle temperature, leaving perfusionable 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 the "activation" of HUVEC cell seeding onto the surrounding collagen. Various other sacrificial materials have also been printed, including “carbohydrate glass” used by Miller et al. as a sacrificial material, which shows the subsequent infusion of hollow networks (Bionic Materials 2012; 11:768-774).

[0006] 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 tissue structures with blood vessels. (Advanced Materials 2014; 26:3124–3130) (Co-printing channel structures of Pluronic F127 and cell-loaded gelatin methacrylate (GelMA); Proceedings of the National Academy of Sciences 2016; 113:3179–3184) (Pluronic F127 mixed with thrombin was designated as "vascular ink" for indirect printing sacrificial channels within cell-loaded gelatin fibrinogen bio-ink). However, it is noteworthy that sacrificial materials such as Pluronic F127 exhibit cytotoxicity at higher concentrations, and it remains unclear what effect liquefied Pluronic would have on the surrounding tissue area, as it is unlikely to be completely removed from the hollow channels.

[0007] A more recent alternative to sacrificing the patterned hollow fiber structure is to use a focused laser beam to heat and ablate regions within the pre-cast (or printed) tissue structure. As the laser beam moves, it leaves a hollow tunnel. This technique can be relatively fast, allowing for 3D patterning of branched hollow tubes with high resolution, potentially with capillaries as small as 10-20 micrometers in diameter. The penetration depth of the beam can be increased using a 2-photon laser, which also helps reduce the intensity of the defocused light, thereby reducing phototoxicity to regions outside the ablation channel.

[0008] Direct bioprinting of hollow tubes within larger tissues has also been explored. For example, Gao et al. demonstrated the ability to generate and print hollow alginate fibers using a calcium chloride crosslinking solution within an alginate fiber core via a coaxial needle. 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 structure with endogenously permeable microchannels. In this approach, hollow microchannels are printed through a gradual descent into a calcium bath solution for secondary crosslinking. (Biomaterials 2015; 61:203–215). Hinton et al. developed an alternative to liquid immersion printing, employing an extrusion method that uses various hydrogels for direct structure printing supported in a sacrificial gelatin-microparticle bath to facilitate crosslinking. (Science Advances 2015; 1:e1500758).

[0009] However, regrettably, the aforementioned systems, devices, and materials used in conventional 3D bioprinting of hollow fiber networks suffer from numerous drawbacks that hinder their more practical, efficient, and widespread implementation. As mentioned above, manually (physically) removing the sacrificial material is impractical, inconsistent, time-consuming, and may be impossible for smaller containers. Furthermore, patterning vascular channels with sacrificial material limits the ability to pattern cells and / or biomaterials in an axial manner around the hollow channel. It is difficult to imagine how this technique could be used to fabricate, for example, patterned channel networks that mimic the structure of real arterioles with smooth muscle cells surrounding the inner lining of endothelial cells.

[0010] Laser ablation limits penetration depth to 1-2 mm and requires optically transparent materials that do not scatter the light beam, while most cellular tissues are opaque and light-scattering. Finally, sacrificial material is extruded and printed, with the diameter of the sacrificial fibers (and subsequently the channel inner diameter) determined by the diameter of the extrusion needle. This diameter is fixed, thus offering no opportunity to dynamically change the lumen diameter of the channels in different regions of the tissue.

[0011] Therefore, there is a need for systems and devices capable of distributing and patterning hollow channels within 3D tissues, wherein pro-angiogenic bio-inks 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 modulated within a single tissue structure, ranging from capillaries to larger vessels. For example, it might be desirable to have a larger diameter vessel at the tissue opening to which a perfusion device is attached, and then reduce the lumen diameter to simulate smaller vessels within the tissue. Changing the vessel diameter could also be a useful tool for modulating blood flow variations and addressing disease limitations such as atherosclerosis. This invention addresses these and other unmet needs. Summary of the Invention

[0012] This invention includes systems and methods for producing core-shell fiber structures comprising hollow fibers and multi-shell structures, as well as for producing three-dimensional (3D) structures from digital files. In some embodiments, the printed fibers comprise living cells. As demonstrated herein, direct printing of core-shell fibers using this invention can produce fibers with different diameters and multiple shells, and different cell types can be loaded into different shells in a precise axial and parallel arrangement to produce hollow blood vessels with multiple cell layers. Furthermore, the composition of the vessel wall (cell type and biomaterial composition) can be modified along the length of the channel during continuous printing.

[0013] An aspect of the invention includes a microfluidic printhead for producing core-shell fiber structures, 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 shell 108; and a dispensing channel 110, wherein the multi-channel shell 108 includes 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 shell 108. In the central region, the core channel outlet 102 is in fluid communication with the inlet of the first fluid focusing chamber 112 and extends to a first vertical depth in the multi-channel housing 108. Preferably, the core channel outlet 102 extends to the first vertical depth in the first fluid focusing chamber 112, aligned with the distribution channel 110. A first housing channel outlet 106 is concentrically disposed around the core channel, in fluid communication with the inlet of the first fluid focusing chamber 112, and extends to a second vertical depth in the multi-channel housing 108. The first fluid focusing chamber 112 converges toward the distribution channel 110. Preferably, the first fluid focusing chamber 112 includes a truncated conical shape configured to focus fluid toward the distribution channel 110. In some embodiments, the first housing channel outlet 106 has a gradient width that increases with depth into the multi-channel housing 108. In an exemplary embodiment, the first housing channel outlet 106 includes a hollow cylinder having an axis of rotation that does not intersect the core channel outlet 102.

[0014] In some embodiments, the core channel outlet 102 extends through a substantial length of the multichannel housing 108. In a preferred embodiment, a first vertical depth is greater than a second vertical depth, such that the core channel outlet 102 extends further into the multichannel housing 108 and / or the first fluid focusing chamber 112 than the first housing channel outlet 106. In an alternative embodiment, a second vertical depth is greater than a first vertical depth, such that the first housing channel outlet 106 extends further into the multichannel housing 108 than the core channel outlet 102. In some embodiments, the first fluid focusing chamber 112 is located on a separate layer of the printhead, and the first housing channel outlet 106 extends from a preceding layer of the printhead into the first fluid focusing chamber 112 in an adjacent downstream layer of the printhead.

[0015] In some embodiments, the printhead includes at least two core channels that converge to form a core channel outlet 102 in fluid communication with the first fluid focusing chamber 112. In a preferred embodiment, at least two core channels converge at or near the core channel outlet 102, the multichannel housing 108, and / or the fluid distribution orifice further described herein. In a particularly preferred embodiment, at least two sub-channels converge in an adjacent preceding layer, or in the same layer of the printhead as the core channel outlet 102, the multichannel housing, and / or the fluid distribution orifice. In one embodiment, the core channels are configured to dispense a non-crosslinkable material. In an exemplary embodiment, the first core channel includes a sheath fluid inlet orifice and a control valve, and the second core channel includes a buffer solution inlet orifice and a control valve.

[0016] In some embodiments, the printhead further includes a second shell channel 128 having at least one inlet and one outlet, wherein the second shell channel outlet and / or inlets 132, 106 are concentrically disposed around the first shell channel outlet 106 in the multi-channel housing 108 within the same layer of the printhead and are 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 preceding layer of the printhead into the first fluid focusing chamber 112 in an adjacent downstream layer of the printhead.

[0017] In an alternative embodiment, the printhead further includes: a second shell channel 128 having at least one inlet and one outlet; and a second multi-channel housing 134 located between the first fluid focusing chamber 112 and the distal end of the dispensing channel 110, wherein the second multi-channel housing 134 includes the dispensing channel 110, a second shell channel outlet 132, and a second fluid focusing chamber 136; wherein the dispensing channel 110 is disposed in the central region of the second multi-channel housing 134, fluidly communicates with the inlet of the second fluid focusing chamber 136, and extends to a first vertical depth in the multi-channel housing, preferably wherein; wherein the second shell channel outlet 132 is concentrically disposed around the dispensing channel 110, fluidly communicates with the inlet of the second fluid focusing chamber 136, and extends to a second vertical depth in the multi-channel housing; and wherein the second fluid focusing chamber 136 converges toward the dispensing channel 110, preferably wherein the second fluid focusing chamber 136 includes a truncated conical shape configured to focus fluid toward the dispensing channel 110. In some embodiments, the first multichannel housing 108 and the second multichannel housing 134 are located in consecutive layers of the printhead. In other embodiments, the first and second multichannel housings, or portions thereof, may be located in the same layer of the printhead; for example, the second multichannel housing may 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 the preceding layer of the printhead into the second fluid focusing chamber 136 in an adjacent downstream layer.

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

[0019] In some embodiments, the first 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, such that the composition of the first and / or second shell layers can vary along the length of the printed fiber. In some embodiments, the first shell channel inlet 104 and / or the second shell channel inlet 130 include different sub-channels with different fluid reservoirs, inlet orifices, and control valves. In some embodiments, the sub-channels have the same fluid reservoir, inlet orifice, and control valve. In other embodiments, the first and / or second shell channels may include two fluid switches, and each sub-channel may be fluidly connected to a different fluid switch. In yet another embodiment, the core channel may include two fluid switches, and each core inlet sub-channel may be fluidly connected to a different fluid switch.

[0020] In one embodiment, the first shell channel inlet 104 and / or the second shell channel inlet 130 include two or more shell channel sub-channels 126, each having a different fluid reservoir, inlet orifice, and control valve, which converge to form a single shell channel outlet 106, 132, and / or fluid distribution orifice. In an exemplary embodiment, a softer hydrogel material flowing through one shell channel sub-channel 126 can be switched to a harder hydrogel material flowing through the second shell channel sub-channel to reinforce the fibers when needed. In another exemplary embodiment, a first cellular material in one shell channel sub-channel 126 can be switched to a second cellular material in the second shell channel sub-channel 126 to generate a range of cell types along the length of the fiber.

[0021] In one embodiment, the first shell channel inlet 104 and / or the second shell channel inlet 130 include three or more shell inlet sub-channels 126, each having a different fluid reservoir, inlet orifice, and control valve, which 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 sub-channels 126 includes a buffer solution inlet orifice and a control valve, and is configured to dispense a buffer to facilitate the displacement of crosslinkable material within the dispensing channel 110.

[0022] In an additional embodiment, 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 converges at a separate fluid distribution orifice connecting the first shell channel inlet 104 and / or the second shell channel inlet 130 to the apex 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.

[0023] In one embodiment, the first shell channel includes at least one fluid distribution orifice connecting the first shell channel inlet 104 to the apex 116 of the upper curved surface 114 of the first shell channel outlet 106, such that 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 sub-channels 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 sub-channels 126 that converge at or near two fluid distribution orifices, preferably located on opposite sides of the first shell channel outlet 106.

[0024] In another embodiment, the second shell channel 128 includes at least one fluid distribution orifice connecting the second shell channel inlet 130 to the apex 116 of the upper curved surface 114 of the second shell channel outlet 132, such that 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.

[0025] In some embodiments, the printhead further includes a sheath flow channel 118 that converges with the distribution channel 110 at a sheath fluid crossover point located between the fluid focusing chamber and the distal end of the distribution channel 110. In some embodiments, the sheath flow channel 118 includes a plurality of sheath flow sub-channels that converge toward the distribution channel 110 via a sheath fluid chamber 120. In a preferred embodiment, the sheath fluid chamber 120 includes a truncated conical shape configured to focus fluid toward the distribution channel 110. In some embodiments, the sheath fluid crossover point is located in the last / final downstream layer of the printhead. In some embodiments, the distribution channel 110 extends from the penultimate layer of the printhead into the sheath fluid chamber 120 in the final downstream layer.

[0026] In one embodiment, the minimum diameter of the truncated cone at the outlets of the fluid focusing chamber and the sheath 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 also includes a dispensing orifice located at the distal end of the dispensing channel 110. In some embodiments, the printhead also includes an extension tip comprising a tube having an exterior configured to fit into a portion of the dispensing channel 110 and an inner surface configured to align with the dispensing channel 110 (defining a hollow space in the tube).

[0027] In one embodiment, the sheath flow channel 118 includes a sheath fluid inlet and a control valve; preferably, the printhead is configured to dispense 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.

[0028] In another aspect, the present invention provides a printhead comprising a plurality of stacked and 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 dispensing 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 dispensing channel 110; wherein the plurality of shell channels are concentrically disposed around the core channel in the same layer of the printhead and 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, and wherein the core channel extends longer into the fluid focusing chamber than any shell channel; and a sheath flow channel 118 converging with the dispensing channel 110 at a sheath fluid crossover point located between the fluid focusing chamber and the distal end of the dispensing channel 110.

[0029] In some embodiments, the printhead further includes a plurality of fluid distribution orifices configured to circumferentially distribute fluid around a plurality of shell channels, wherein each fluid distribution orifice individually connects a corresponding shell channel inlet 122 to the 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 a second shell channel outlet 132 has a parabolic or elliptical shape. In an exemplary embodiment, at least one of the plurality of shell channels has a gradient width that increases with increasing longitudinal depth into the shell.

[0030] In some embodiments, the printhead further includes third, fourth, fifth, and / or sixth shell channels having at least one inlet and one outlet, wherein each of the third, fourth, fifth, and / or sixth shell channel outlets is concentrically disposed around an adjacent preceding shell channel outlet in a multi-channel shell within 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 outlet extends 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 shell than the adjacent preceding shell channel outlet, and the core channel extends further into the multi-channel shell than the first shell channel.

[0031] In an alternative embodiment, the printhead further includes: third, fourth, fifth, and / or sixth shell channels, each shell channel having at least one inlet and outlet; and third, fourth, fifth, and / or sixth multi-channel shells located between the second fluid focusing chamber 136 and the distal end of the dispensing channel 110, wherein the third, fourth, fifth, and / or sixth multi-channel shells include the dispensing channel 110, the outlets of the third, fourth, fifth, and / or sixth shell channels, and the third, fourth, fifth, and / or sixth fluid focusing chambers; wherein the dispensing channel 110 is disposed in the central region of the respective multi-channel shell, and... The inlet of the respective fluid focusing chamber is in fluid communication and extends to a first vertical depth in the multi-channel housing, wherein the third, fourth, fifth, and / or sixth housing channel outlets are concentrically disposed around the distribution channel 110 and in fluid communication with the inlet of the respective fluid focusing chamber, extending to a second vertical depth in the respective 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 truncated conical shape configured to focus fluid toward the distribution channel 110. In some embodiments, the third, fourth, fifth, and / or sixth multi-channel housings are located on a continuous layer of the printhead. In some embodiments, the housing channel outlets may extend from a previous layer of the printhead to a respective fluid focusing chamber in an adjacent downstream layer of the printhead.

[0032] 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 including at least two core inlet sub-channels having different fluid reservoirs, inlet orifices, and control valves, which converge to form a single core channel outlet 102 in fluid communication with a first fluid focusing chamber 112; a first shell channel including at least two shell inlet sub-channels 126 having different fluid reservoirs, inlet orifices, and control valves, which 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 truncated conical shape configured to focus fluid toward the distribution channel 110; and a sheath flow channel 118 which converges with the distribution channel 110 at a sheath fluid crossover point located between the second fluid focusing crossover point and the distal end of the distribution channel 110. In some embodiments, at least two core inlet sub-channels converge at or near core channel outlet 102, and preferably converge in the preceding layer of the printhead that is the same as or adjacent to core channel outlet 102. In some embodiments, the first shell channel includes three shell inlet sub-channels 126, one of which is connected to a fluid reservoir containing a buffer solution.

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

[0034] An aspect of the invention includes a system for producing fiber structures, the system comprising a printhead including: a core channel having an inlet and an outlet; a first shell channel having an inlet and an outlet; a multi-channel shell; and a dispensing channel 110, wherein the multi-channel shell includes 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 shell, in fluid communication with the inlet of the fluid focusing chamber, and extends to a first vertical depth in the multi-channel shell, preferably wherein the core channel outlet 102 extends to the first vertical depth in the fluid focusing chamber and is aligned with the dispensing channel 110; wherein the first shell channel... Outlet 106 is concentrically disposed around the core channel, in fluid communication with the inlet of the fluid focusing chamber, and extends to a second vertical depth in the multi-channel housing; 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 fluid toward the distribution channel 110; a sheath flow channel 118, which converges with the distribution channel 110 at a sheath fluid crossover point located between the first fluid focusing crossover point and the 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 operatively coupled to the printhead.

[0035] In some embodiments, the system further includes 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 includes a fluid removal assembly configured to remove excess fluid dispensed from the printhead. In some embodiments, the fluid removal assembly includes a porous membrane configured to allow excess fluid to pass through. In some embodiments, the fluid removal assembly includes an absorbent material. In some embodiments, the fluid removal assembly includes a vacuum configured to aspirate excess fluid. In some embodiments, a vacuum is applied below the receiving surface. In some embodiments, a vacuum is applied above the receiving surface. In some embodiments, a vacuum is applied through one or more vacuum channels on the printhead. In some embodiments, one or more vacuum channels are positioned near a dispensing orifice on the printhead.

[0036] In some embodiments, the system further includes a pressure control component configured to regulate the flow rate of one or more fluids through the printhead. In some embodiments, the system further includes one or more fluid reservoirs in fluid communication with the printhead. In some embodiments, the fluid reservoir includes a sheath solution. In some embodiments, the sheath solution includes a crosslinking solution configured to cure an input material. In some embodiments, the crosslinking solution includes a divalent cation. In some embodiments, the divalent cation is Ca++. In some embodiments, the fluid reservoir includes a buffer solution. In some embodiments, the buffer solution is miscible with the input material. In some embodiments, the fluid reservoir includes the input material. In some embodiments, the input material includes a crosslinkable material, such as a hydrogel. In some embodiments, the hydrogel comprises alginate. In some embodiments, the alginate is depolymerized alginate. In some embodiments, the input material includes one or more living cells. In some embodiments, the input material includes an extracellular matrix material. In some embodiments, the input material includes a surfactant.

[0037] In some embodiments, the system further includes a printhead comprising at least two shell inlet sub-channels 126 and / or a first shell channel, the first shell channel comprising at least two shell channels connected to a fluid reservoir comprising different input materials, and the method includes generating a core-shell fiber structure comprising a first input material and a second input material. In some embodiments, the method includes dispensing 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 includes dispensing first and second input materials through the shell inlet sub-channels 126 to generate a cured fiber structure comprising different shell materials along the length of a continuous fiber structure.

[0038] In some embodiments, the printhead is configured to generate a constant mass flow rate through the dispensing channel 110. In some embodiments, the system also includes a crosslinking assembly. In some embodiments, the crosslinking assembly includes a UV lamp. In some embodiments, the crosslinking assembly is located near the dispensing orifice.

[0039] An aspect of the invention includes a method for producing a cured fiber structure, the method comprising: providing a system for producing the fiber structure, the system including: a printhead comprising a plurality of stacked and preferably bonded layers forming a plurality of fluid channels, the printhead including 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 dispensing channel 110, wherein the multi-channel housing 108 includes 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, in fluid communication with the inlet of the first fluid focusing chamber 112, and extends to a first vertical depth in the multi-channel housing 108, preferably wherein the core channel outlet 102 extends to the first vertical depth in the fluid focusing chamber and is aligned with the dispensing channel 110; wherein the first shell channel outlet 106 is concentrically disposed around the core channel, in fluid communication with the inlet of the fluid focusing chamber, and extends to a second vertical depth in the multi-channel housing 108; and wherein the fluid focusing chamber converges toward the dispensing channel 110. Preferably, the fluid focusing chamber includes a truncated conical shape configured to focus fluid toward the dispensing channel 110; a sheath flow channel 118 converging with the dispensing channel 110 at a sheath fluid crossover point located between the first fluid focusing crossover point and the distal end of the dispensing channel 110; a receiving surface for receiving a first layer of material dispensed from the printhead; a positioning assembly for positioning a dispensing orifice of the printhead in 3D space, wherein the positioning assembly is operatively coupled to the printhead; a programmable control processor for controlling the positioning assembly and for controlling the flow rate of one or more fluids through the printhead; a first fluid reservoir including a first input material; a second fluid reservoir including a buffer solution; and a third fluid reservoir including a sheath solution, wherein the sheath solution includes a crosslinking solution; wherein the fluid reservoirs are in fluid communication with the printhead; the first input material is passed through the dispensing channel 110; the first input material is crosslinked with a crosslinking assembly to produce a cured fiber structure; and the cured fiber structure is dispensed from the dispensing orifice of the printhead.

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

[0041] In some embodiments, the non-crosslinkable material in the core channel comprises a buffer solution and the sheath fluid in the sheath flow channel 118 comprises a chemical crosslinking agent, and contact occurs at the sheath fluid crossover point to solidify the outer surface of the crosslinkable material flow in the distribution channel 110.

[0042] In some embodiments, the non-crosslinkable material in the core channel comprises a chemical crosslinking agent 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.

[0043] In some embodiments, the non-crosslinkable material in the core channel includes a chemical crosslinking agent, and the sheath fluid in the sheath flow channel 118 includes a chemical crosslinking agent, 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.

[0044] In some implementations, one method further includes: encoding a programmable control processor with the planar structure to be printed; and depositing a first layer of a cured fiber structure onto a receiving surface to print the planar structure.

[0045] In some implementations, one method further includes: encoding a programmable control processor with the 3D structure to be printed; and depositing subsequent layers of a cured fiber structure on top of a planar structure to print the 3D structure.

[0046] In another embodiment, the present invention provides a method for producing a continuously cured fiber structure having a core and / or shell component varying along the fiber length, the method comprising: providing a system for producing the fiber structure, the system comprising: a printhead including a core channel, the core channel including at least two core inlet sub-channels, the core inlet sub-channels having different fluid reservoirs, inlet orifices, and control valves converging to form a single core channel outlet 102 in fluid communication with a first fluid focusing chamber 112; and a first shell channel including at least two shell inlet sub-channels 126. The shell inlet sub-channel has different fluid reservoirs, inlet orifices, and control valves that converge to form a single shell channel outlet in fluid communication with the second fluid focusing chamber 136; and a distribution channel 110; wherein the fluid focusing chamber converges toward the distribution channel 110, preferably wherein the fluid focusing chamber includes a truncated 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 crossover point located between the second fluid focusing crossover point and the distal end of the distribution channel 110; and a receiving surface for receiving fluid dispensed from the printhead. First layer material; a positioning assembly for positioning the dispensing orifice of the printhead in 3D space, wherein the positioning assembly is operatively coupled to the printhead; a programmable control processor for controlling the positioning assembly and for controlling the flow rate of one or more fluids through the printhead; a first fluid reservoir including a first input material connected to a first core inlet subchannel; a second fluid reservoir including a second input material connected to a second core inlet subchannel; a third fluid reservoir including a third input material connected to a first shell inlet subchannel 126; a fourth A fluid reservoir, the fourth fluid reservoir including a fourth input material connected to a second shell inlet sub-channel 126; a fifth fluid reservoir including a sheath solution connected to a sheath flow channel 118, wherein the sheath solution includes a crosslinking solution; wherein the fluid reservoirs are in fluid communication with a printhead; a first or second input material is alternately passed through a dispensing channel 110; and simultaneously, a third or fourth input material is alternately passed through the dispensing channel 110, such that the first, second, third, and / or fourth input materials are crosslinked with a crosslinking assembly to produce a cured fiber structure; and the cured fiber structure is dispensed from a dispensing orifice of the printhead. In some embodiments, the first and / or second input materials include a non-crosslinkable material.

[0047] In another embodiment, the first shell channel includes three shell inlet sub-channels, and a sixth fluid reservoir including a buffer solution is connected to the third shell inlet sub-channel 126, and the method includes passing the buffer solution through the dispensing channel 110 to displace the crosslinkable material and terminate the fiber.

[0048] On the other hand, it is also expected that bioprinted tissue fibers produced by thematic methods will have variable core and shell materials along the entire length of the fiber.

[0049] This invention also successfully addresses the technical challenges of manufacturing synthetic perfusionable hollow tissue fibers capable of attaching to an external perfusion system without breaking, as detailed and illustrated in Example 1 herein. In one aspect, the invention provides a bioprinted tissue fiber comprising: an inner lumen; a continuous inner shell surrounding the lumen, the inner shell including reinforcing hydrogel materials at distal and proximal ends of the fiber; and a biocompatible hydrogel material located therebetween. The biocompatible hydrogel material preferably comprises at least one biomaterial, such as living cells, while the reinforcing hydrogel material is cell-free. In another embodiment, the fiber further includes a second continuous outer shell comprising the reinforcing hydrogel material.

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

[0051] In some embodiments, at least one biological material 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 gland (follicular cells), pineal gland (pineal cells), pituitary gland (growth hormone, prolactin, gonadotropins, adrenocorticotropic hormone, and thyroid-stimulating hormone), thymus (thymic cells, 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 a preferred embodiment, at least one biological material comprises a cell population expressing / secreting bioactive agents, such as insulin, glucagon, auxin, pancreatic polypeptide, angiogenic factors, growth factors, hormones, antibodies, enzymes, proteins, exosomes, etc.

[0052] The invention also includes a method for producing perfusable hollow tissue fibers, the method comprising: providing a printhead according to the invention, the printhead including a first shell channel including at least two shell inlet sub-channels 126, the shell inlet sub-channels having different fluid reservoirs, inlet orifices, and control valves; dispensing sheath fluid through a core channel, a reinforcing hydrogel material through the first shell inlet sub-channel 126, a biocompatible hydrogel material comprising one or more biomaterials through the second shell inlet sub-channel 126, and sheath fluid through a 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 at the end of the perfusable fiber to allow attachment to an external perfuscation system, for example, by needle insertion. In another embodiment, the printhead further includes a second shell channel 128 as described herein, and the method further includes dispensing the same or different reinforcing hydrogel materials through the second shell channel 128 to create a concentric second shell surrounding the first shell material, and further reinforcing the fiber and preventing breakage along the entire length of the fiber.

[0053] This invention also successfully addresses the technical challenges of manufacturing synthetic perfusionable hollow tissue fibers capable of attaching to an external perfusion system without breaking, as detailed and illustrated in Example 1 herein. In one aspect, the invention provides a bioprinted tissue fiber comprising: an inner lumen; a continuous inner shell surrounding the lumen, the inner shell including reinforcing hydrogel materials at distal and proximal ends of the fiber; and a biocompatible hydrogel material located therebetween. The biocompatible hydrogel material preferably comprises at least one biomaterial, such as living cells, while the reinforcing hydrogel material is cell-free. In another embodiment, the fiber further includes a second continuous outer shell comprising the reinforcing hydrogel material. Attached Figure Description

[0054] Figure 1 This is an illustration of the concentric shell printhead design with a single fluid distribution orifice of the present invention.

[0055] Figure 2 A side view and close-up detail are provided of the multi-channel housing and sheath flow chamber in the concentric housing printhead design of the present invention, which has a single fluid distribution orifice.

[0056] Figure 3 The key components of the microfluidic pathway in the concentric shell printhead design of the present invention, which has a single fluid distribution orifice, are shown and identified.

[0057] Figure 4AThis is a diagram illustrating the flow pattern of a single fluid distribution orifice in the concentric shell printhead design of the present invention. Figure 4B This is a diagram illustrating the flow pattern of a single fluid distribution orifice in a multi-channel shell and sheath flow chamber designed by the concentric shell printhead of the present invention.

[0058] Figure 5 A side view and close-up detail are provided of the multi-channel housing and sheath flow chamber in the multi-shell concentric printhead design of the present invention, which has two fluid inlet orifices.

[0059] Figure 6 The key components of the microfluidic pathway in the multi-shell concentric printhead design of the present invention, which has two fluid distribution orifices, are shown and identified.

[0060] Figure 7A This is an illustration of the flow pattern of the two fluid distribution orifices in the multi-shell printhead design of the present invention, which has two fluid distribution orifices. Figure 7B This is a diagram illustrating the flow pattern of the multi-channel outer shell and two fluid distribution orifices in the sheath flow chamber of the multi-shell printhead design of this invention.

[0061] Figure 8A and Figure 8B The illustrations and close-up details of embodiments of the invention are provided, which include two distinct shell inlet sub-channels converging at a single fluid distribution orifice.

[0062] Figures 9A to 9C A transparent view (10A), a top view (10B), and an exploded view (10D) of a printhead design according to the invention are provided, the printhead design including three distinct shell inlet sub-channels converging at a single fluid distribution orifice.

[0063] Figures 10A to 10C A transparent view (10A), a top view (10B), and an exploded layer view (10C) of a printhead design according to the present invention are provided, the printhead design including a second shell channel, a second multi-channel shell, and a second fluid focusing chamber in a layer of the printhead different from the first fluid focusing chamber.

[0064] Figure 11 A transparent view of a printhead design according to the present invention is provided, the printhead design including a second shell channel, a second multi-channel shell, and a second fluid focusing chamber, wherein the second multi-channel shell overlaps with the first fluid focusing chamber in the same layer of the printhead.

[0065] Figure 12 Various illustrations of infusable hollow tissue fibers according to the present invention are provided.

[0066] Figure 13Additional illustrations of the infusible hollow tissue fibers according to the present invention are provided.

[0067] Figure 14 Various illustrations of synthetic tissue fibers according to the invention are provided, the synthetic tissue fibers comprising a core and / or shell composition that varies along the fiber length.

[0068] Figure 15 A synthetic hollow fiber according to the present invention is provided, the synthetic hollow fiber having a certain range of internal lumens and fiber diameters. Detailed Implementation

[0069] The present invention includes systems and methods for producing core-shell fiber structures comprising hollow core fibers and multi-shell fibers, and for producing three-dimensional (3D) structures from digital files. In some embodiments, the printed fibers comprise living cells.

[0070] definition:

[0071] For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form and vice versa. In the event of any conflict between any definition set forth and any document incorporated herein by reference, the definition shown below shall prevail.

[0072] As used herein, the term "displacement" refers to the ability of a first material or fluid to remove a second material or fluid from a given location. For example, in some embodiments, a buffer solution is configured to displace input material from a location within dispensing channel 110 (e.g., from the proximal end of dispensing channel 110). In some embodiments, the displacement is instantaneous, occurring in less than about one second, such as in about 900, 800, 700, 600, 500, 400, 300, 200, or 100 milliseconds or less.

[0073] As used in this article, the term “miscible” refers to the ability of two different liquids to form a homogeneous mixture when mixed.

[0074] As used herein, the term "mass flow rate" refers to the mass of 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.

[0075] As used herein, the term "cured" refers to a solid or semi-solid material that retains its shape fidelity and structural integrity upon deposition. As used herein, the term "shape fidelity" refers to the ability of a material to retain its three-dimensional shape without significant diffusion. In some embodiments, the cured material is a material capable of retaining its three-dimensional shape for a time 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 loads including its own weight while resisting fracture or bending.

[0076] In some embodiments, the cured composition has an elastic modulus greater than about 5, 10, 15, 20, or 25 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 invention, the elastic modulus of the input material can be advantageously varied 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.

[0077] As used herein, the term “natural alginate polymer” refers to an alginate polymer that has been isolated and purified from one or more natural sources (e.g., one or more brown algae or seagrasses).

[0078] As used in this article, the term “depolymerization” refers to breaking down a polymer chain into monomers or other smaller units.

[0079] As used herein, the term "hydrogel" refers to a composition comprising a network or lattice of water and hydrophilic polymer chains.

[0080] As used herein, the terms "sheath fluid" or "sheath solution" refer to a fluid used to at least partially encapsulate or "wrap" a material as it passes through a fluid channel. In some embodiments, the sheath fluid comprises an aqueous solvent, such as water or glycerol. In some embodiments, the sheath fluid comprises a chemical crosslinking agent. Non-limiting examples of crosslinking agents include divalent cations (e.g., Ca2+). 2+ Ba 2+ 、Sr 2+ (etc.), thrombin, and pH-modifying chemicals, such as sodium bicarbonate.

[0081] As used herein, the term "excess sheath fluid" refers to a portion of sheath fluid dispensed from a dispensing orifice that does not form part of a fibrous structure printed using one or more embodiments of the system or method provided herein. For example, excess sheath fluid may be used to lubricate material (e.g., hydrogel) through dispensing channels 110 in the printhead and through channels in 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.

[0082] As used in this article, the term "channel length" refers to the linear distance traveled when tracing a fluid channel from a first position to a second position.

[0083] As used in this article, the term "convergence angle" refers to the angle formed between two converging fluid channels.

[0084] Print head:

[0085] Aspects of the invention include a printhead capable of producing one or more core-shell fiber structures, the core-shell fiber structures comprising multi-shell fibers and / or hollow fibers. A printhead according to an embodiment of the invention comprises a plurality of stacked layers forming a plurality of interconnected fluid channels flowing vertically through the layers, these layers preferably being bonded 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 printhead is configured to produce a cured hollow fiber structure. In some embodiments, the printhead is configured to produce a cured hollow fiber structure comprising living cells.

[0086] In some embodiments, the printhead includes a dispensing channel 110 having a distal end and a proximal end. The dispensing channel according to embodiments of the invention may 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. The dispensing channel according to embodiments of the invention may 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 dispensing channel according to embodiments 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 dispensing channel according to embodiments of the present invention can have any suitable cross-sectional shape, such as circular, elliptical, square or rectangular cross-sectional shapes.

[0087] In some embodiments, the dispensing channel 110 includes a dispensing orifice. In some embodiments, the dispensing orifice is located at the distal end of the dispensing channel 110. The dispensing orifice according to embodiments of the invention may 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 dispensing orifice according to embodiments of the invention may have any suitable cross-sectional shape, such as circular, elliptical, square, or rectangular cross-sectional shapes.

[0088] In some embodiments, the printhead further includes an extension tip comprising an orifice for dispensing material from the printhead. This extension tip facilitates precise material dispensing and deposition in confined areas, such as the pores in a multi-well plate (e.g., a standard microtiter plate, a multi-well plate, or a microplate with 6, 24, 96, or more pores) or a culture dish. In some embodiments, the extension tip comprises a tube (e.g., made of plastic, glass, or metal) having an exterior configured to fit into a portion of a dispensing channel 110 and an inner surface configured to align with the dispensing channel (defining a hollow space within the tube). The extension tip can be inserted into the dispensing channel 110, thereby extending the length of the dispensing channel 110, which facilitates the deposition of material dispensed from the orifice in the extension tip into the confined space of, for example, a well plate insert or a culture dish.

[0089] A printhead according to an embodiment of the invention includes one or more core channels. In some embodiments, the one or more core channels converge with the dispensing channel 110 at the proximal end of the dispensing channel 110. In some embodiments, the core channels converge with the dispensing channel 110 at a convergence angle ranging 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 core channels according to an embodiment of the 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 invention may 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 material channel according to embodiments of the present invention may 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.

[0090] In some embodiments, the printhead includes at least two core channels having the same or different fluid reservoirs, inlet orifices, and control valves, which converge to form a single core channel outlet 102 in fluid communication with the fluid focusing chamber. In a preferred embodiment, at least two core channels converge at or near the core channel outlet 102, the multichannel housing 108, and / or the fluid distribution orifice further described herein, for example, within a range of about 100 μm to about 50 mm, thereby reducing travel distance and bringing the material transition point closer to the curing point. The inventors have determined that this can prevent tailing between material transitions within the printed fibers. In a particularly preferred embodiment, at least two sub-channels converge in an adjacent preceding layer, or in the same layer of the printhead as the core channel outlet 102, the multichannel housing, and / or the fluid distribution orifice. In some embodiments, the channel length between the convergence point of the sub-channels and the core channel outlet 102, the multi-channel 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 printhead includes a plurality of core sub-channels ranging from 3 to 10, for example, 4, 5, 6, 7, 8, or 9 sub-channels. The core channels according to embodiments of the invention can have any suitable cross-sectional shape, such as circular, elliptical, square, or rectangular cross-sectional shapes. In some embodiments, the printhead is configured to dispense non-crosslinkable material through the core channels.

[0091] A printhead according to an embodiment of the present invention includes: a core channel having an inlet and an outlet; a first shell channel having an inlet and an outlet; a multi-channel shell 108; and a dispensing channel 110, wherein the multi-channel shell 108 includes 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 shell 108, fluidly communicating with the inlet of the fluid focusing chamber, and extends to a first vertical depth in the multi-channel shell 108, preferably wherein the core channel outlet 102 extends to the first vertical depth in the fluid focusing chamber and is aligned with the dispensing channel 110; wherein the first shell channel outlet 106 is concentrically disposed around the core channel, fluidly communicating with the inlet of the fluid focusing chamber, and extends to a second vertical depth in the multi-channel shell 108; and wherein the fluid focusing chamber converges toward the dispensing channel 110, preferably wherein the fluid focusing chamber includes a truncated conical shape configured to focus fluid toward the dispensing channel 110. In some embodiments, the first shell channel outlet 106 has a gradient width that increases with depth into the multi-channel shell 108. In an exemplary embodiment, the first shell channel outlet 106 comprises a hollow cylinder having an axis of rotation that does not intersect with the core channel outlet 102.

[0092] In some embodiments, the core channel outlet 102 extends through a significant portion of the length of the multichannel housing 108. In a preferred embodiment, the first vertical depth is greater than the second vertical depth, such that the core channel outlet 102 extends further into the multichannel housing 108 and / or the first fluid focusing chamber 112 than the first housing channel outlet 106. In an alternative embodiment, the second vertical depth is greater than the first vertical depth, such that the first housing channel outlet 106 extends further into the multichannel housing 108 than the core channel outlet 102.

[0093] In some embodiments, the printing process further includes a second shell channel 128 having an inlet and an outlet, wherein the second shell channel outlet 132 and / or inlet 130 are concentrically disposed around the first shell channel outlet 106 in the same layer of the printhead and are in fluid communication with the 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 second shell channel inlet 130 may be adjacent to the first shell channel outlet 106 in the same layer of the printhead and may be in fluid communication with the first fluid focusing chamber 112.

[0094] In an alternative embodiment, the printhead further includes: a second shell channel having at least one inlet and one outlet; and a second multi-channel housing located between the distal end of the first fluid focusing chamber and the dispensing channel, wherein the second multi-channel housing includes the dispensing channel, the outlet of the second shell channel, and the second fluid focusing chamber; wherein the dispensing channel is disposed in a central region of the second multi-channel housing, in fluid communication with the inlet of the second fluid focusing chamber, and extends to a first vertical depth in the multi-channel housing; preferably, the outlet of the second shell channel is concentrically disposed around the dispensing channel, in fluid communication with the inlet of the second fluid focusing chamber, and extends to a second vertical depth in the multi-channel housing; and wherein the second fluid focusing chamber converges toward the dispensing channel, preferably wherein the second fluid focusing chamber includes a truncated conical shape configured to focus fluid toward the dispensing channel. In some embodiments, the first and second multi-channel housings, or portions thereof, are located in the same layer of the printhead, for example, as... Figure 11 As shown, the second multichannel housing may overlap with the first fluid focusing chamber. In some embodiments, the first and second multichannel housings are located in a continuous layer of the printhead, for example, as... Figure 10A As shown in the diagram. 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 the previous layer of the printhead into the second fluid focusing chamber in the adjacent downstream layer.

[0095] The core and shell channels according to embodiments of the invention can have any suitable length. In some embodiments, the core or shell 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 and shell channels according to embodiments of the 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 core and shell channels according to embodiments 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 core and shell channels according to embodiments of the present invention can have any suitable cross-sectional shape, such as circular, elliptical, square or rectangular cross-sectional shapes.

[0096] In some embodiments, the first shell channel, the second shell channel 128, or both further include at least one fluid distribution orifice configured to circumferentially distribute fluid around the first shell channel outlet 106 and / or the second shell channel outlet 132. Preferably, the fluid distribution orifice connects the first shell channel inlet 104 and / or the second shell channel inlet 130 to the apex 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 channels include at least two shell sub-channels that may converge at a single fluid distribution orifice or lead to separate fluid distribution orifices. The shell channels may be fluidly connected to the same fluid reservoir, inlet port, and control valve, or fluidly connected to separate fluid reservoirs, inlet ports, and control valves. In some embodiments, the printhead includes a plurality of shell channels ranging from 3 to 10, such as 4, 5, 6, 7, 8, or 9 shell channels. The shell channel and sub-channel according to embodiments of the present invention can have any suitable cross-sectional shape, such as circular, elliptical, square or rectangular cross-sectional shapes.

[0097] In an additional embodiment, 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 includes a separate fluid distribution orifice connecting the first shell channel inlet 104 and / or the second shell channel inlet 130 to the apex 116 of the upper curved surface 114 of the first shell channel outlet 106 and / or the second shell channel outlet 132. Preferably, 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 inlet orifice is 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.

[0098] In one embodiment, the first shell channel includes at least one fluid distribution orifice connecting the first shell channel inlet 104 to the apex 116 of the upper curved surface 114 of the first shell channel outlet 106, such that 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 to the apex 116 of the upper curved surface 114 of the second shell channel outlet 132, such that 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.

[0099] A printhead according to an embodiment of the invention includes a sheath flow channel 118. In some embodiments, the sheath flow channel 118 converges with the distribution channel 110 at a sheath fluid crossover point located between a first fluid focusing crossover 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, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, or 175 degrees. In some embodiments, the distance between the proximal end of the dispensing channel 110 and the sheath fluid crossover point ranges from about 10 μm to about 100 mm, for example, 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 dispensing channel 110 and the sheath fluid crossover point ranges from about 10 μm to about 100 mm, for example, 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.

[0100] The sheath flow channel according to embodiments of the present invention can have any suitable length. 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. The sheath flow 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. The sheath flow channel according to embodiments of the 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 branches 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 fluid crossover point. The sheath flow channel according to embodiments of the invention can have any suitable cross-sectional shape, such as circular, elliptical, square, or rectangular cross-sectional shapes.

[0101] The fluid channel according to embodiments of the invention typically includes one or more inlet ports through which fluid can be introduced into the channel, these inlet ports typically being 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, the fluid channel typically being located in the second layer from the top of the printhead stack. In some embodiments, the channel length between the inlet port 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 implementations, the length of the channel between the control valve and the location where the channel and the distribution channel 110 converge 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.

[0102] The printhead according to embodiments of the invention can be made of any suitable material, including but not limited to plastics (e.g., polymer materials), glass, metals, ceramics, or any combination thereof. In a preferred embodiment, the printhead is manufactured using known microfluidic molding techniques (e.g., casting, embossing, or injection molding) and one or more moldable polymers such as polydimethylsiloxane (PDMS), polycarbonate (PC), cyclic olefin polymers (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 printhead.

[0103] In some embodiments, the printhead includes 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 some embodiments, a portion of the printhead surrounding or directly adjacent to the dispensing channel 110 includes a material that is partially or completely transparent to light. Such printheads can be used in conjunction with input materials configured to crosslink with light energy (e.g., photocrosslinkable input materials).

[0104] Aspects of the invention include an optical module configured to expose photocrosslinkable input material to electromagnetic radiation to cause the input material to crosslink. The optical module according to embodiments of the invention may be integrated into a printhead or may be a separate component of the printing system. In some embodiments, the optical module exposes the input material to light while it is within the dispensing channel 110. In some embodiments, the optical module exposes the input material to light after it has been dispensed from the dispensing channel 110. In some embodiments, the printhead includes a plurality of optical modules, wherein a first optical module is configured to expose the input material to light while it is within the dispensing channel 110, and a second optical module is configured to expose the input material to light after it has been dispensed from the dispensing channel 110.

[0105] In some embodiments, the optical module is tunable with respect to wavelength, intensity, exposure time, or any combination thereof. In some embodiments, the optical module includes one or more optionally coupled attenuation filters, wherein the attenuation filters adjust the light intensity when coupled. In some embodiments, the optical 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, such as 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, by way of non-limiting example, suitable UV light sources include UV lamps, UV fluorescent lamps, UV LEDs, UV lasers, or any combination thereof.

[0106] As described above, aspects of the present invention include a printhead comprising a dispensing channel 110, wherein one or more material channels and optionally a buffer solution channel converge at a proximal end of the dispensing channel 110. The printhead of the present invention is configured to simultaneously dispense a buffer solution and / or sheath fluid with one or more crosslinkable 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 dispensing channel 110. In this way, 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 the buffer solution and / or sheath fluid through the dispensing channel 110.

[0107] As described above, an additional aspect of the invention includes a printhead comprising a dispensing channel 110, wherein one or more sheath flow channels 118 converge with the dispensing channel 110 at a sheath fluid crossover point located between a first fluid focusing crossover point and the distal end of the dispensing channel 110. When using the printhead of the invention, input material flowing through the dispensing channel 110 can be crosslinked from the inside by sheath fluid flowing through the core channel and from the outside by sheath fluid flowing through the sheath flow channels 118.

[0108] In a preferred embodiment, the present invention provides a printhead comprising a plurality of stacked and 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 dispensing 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 dispensing channel 110; wherein the plurality of shell channels are concentrically disposed around the core channel in the 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, and wherein the core channel extends longer into the fluid focusing chamber than any shell channel; and a sheath flow channel 118 converging with the dispensing channel 110 at a sheath fluid crossover point located between the fluid focusing chamber and the distal end of the dispensing channel 110.

[0109] In some embodiments, the printhead further includes a plurality of fluid distribution orifices configured to circumferentially distribute fluid around a plurality of shell channels, wherein each fluid distribution orifice individually connects a corresponding shell channel inlet 122 to the 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 of the plurality of shell channels has a gradient width that increases with increasing longitudinal depth into the shell.

[0110] In some embodiments, the printhead further includes third, fourth, fifth, and / or sixth shell channels having inlets and outlets, wherein each of the third, fourth, fifth, and / or sixth shell channel outlets is concentrically disposed around the outlet of the immediately preceding shell channel in the multi-channel shell 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 shell than the outlet of the immediately preceding shell channel, and the core channel extends further into the multi-channel shell than the first shell channel.

[0111] 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 including at least two core inlet sub-channels, the core inlet sub-channels having different fluid reservoirs, inlet orifices and control valves, which 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 sub-channels converge at or near the core channel outlet 102; and a first shell channel including at least two shell inlet sub-channels. 126, the shell inlet sub-channel has different fluid reservoirs, inlet orifices, and control valves that converge to form a single shell channel outlet in fluid communication with the second fluid focusing chamber 136; a distribution channel 110; 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 fluid toward the distribution channel 110; and a sheath flow channel 118 that converges with the distribution channel 110 at a sheath fluid crossover point located between the second fluid focusing crossover point and the distal end of the distribution channel 110. In another embodiment, the first shell channel includes three shell inlet sub-channels 126, one of which is connected to a fluid reservoir comprising a buffer solution.

[0112] In some embodiments, the core channel further includes at least one fluid distribution orifice configured to distribute fluid around the circumference of the core channel outlet 102; preferably, the at least one fluid distribution orifice connects the converging core channel inlet to the apex 116 of the upper curved surface 114 of the core channel outlet 102; more preferably, the upper curved surface 114 has a parabolic or elliptical shape.

[0113] Printing system:

[0114] Aspects of the present invention include a printing system and associated components configured to work with the printhead of the present invention to perform the methods 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 individual printheads, as described herein. In some embodiments, the printhead is fluidly isolated from the printing system such that all fluids involved in the printing process remain isolated within the printhead and contact only the receiving surface of the printing system (described below) during the printing process. In some embodiments, the printhead is configured to be operatively coupled to the printing system without allowing fluids involved in the printing process to contact the components of the printing system. In some embodiments, one or more printheads may be removed and / or one or more printheads may be added to the printing system before, during, and / or after the printing process. Thus, in some embodiments, the printhead of the present invention is a modular component of the printing system of the present invention.

[0115] In some embodiments, the printing system includes a receiving surface on which a first layer of material, dispensed from a dispensing orifice of a printhead, 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 through it. 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 morphology corresponding to the three-dimensional structure to be printed, thereby facilitating the printing of a three-dimensional structure with a non-planar first layer.

[0116] 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 the printing process.

[0117] In some embodiments, the receiving surface is a non-cytotoxic surface on which the printing system distributes one or more fiber structures. In some embodiments, the printing system includes a printing stage. In some embodiments, the receiving surface is the surface of the printing stage. In some embodiments, the receiving surface is a component separate from but attached to or supported by the printing stage. In some embodiments, the receiving surface is flat or substantially flat. In some embodiments, the receiving surface is smooth or substantially smooth. In some embodiments, the receiving surface is substantially flat and substantially smooth. In some embodiments, the receiving surface is configured to adapt to 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.

[0118] In some embodiments, the receiving surface includes one or more modular components configured to be operatively coupled to the printing system but detachable from it. 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.

[0119] In some embodiments, the receiving surface is configured to be operatively coupled to one or more different receiving containers. For example, in some embodiments, the receiving surface includes a circular portion sized to be operatively coupled to a circular receiving container (e.g., a petri dish). In some embodiments, the receiving surface includes a square or rectangular portion sized to be operatively coupled to a square or rectangular receiving container (e.g., a multi-well plate (e.g., a 6-well plate)). The receiving surface according to embodiments of the invention can have any suitable size or geometry to accommodate a suitable receiving container.

[0120] In some embodiments, the printing system includes a temperature regulation assembly configured to regulate the temperature of a receiving surface. In some embodiments, the temperature regulation assembly adjusts and / or maintains the temperature of the receiving surface at ambient temperature. In some embodiments, the temperature regulation assembly adjusts and / or maintains the temperature of the printhead, print stage, receiving surface, input material and / or fluid (e.g., sheath solution and / or buffer solution).

[0121] 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 chilled 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.

[0122] In some implementations, the temperature regulating component is configured to regulate the temperature to a setpoint ranging from about 0 to about 90°C, such as about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85°C.

[0123] In some embodiments, the printing system achieves a specific geometry by moving the printhead relative to a print stage or a receiving surface adapted to receive printing material. In other embodiments, the printing system achieves a specific geometry by moving the print stage or receiving surface relative to the printhead. In some embodiments, at least a portion of the printing system is maintained in a sterile environment (e.g., within a biosafety cabinet (BSC)). In some embodiments, the printing system is configured to be fully adapted to a sterile environment.

[0124] In some embodiments, the receiving surface receives excess fluid (e.g., excess sheath fluid and / or excess buffer solution), which is dispensed from a dispensing orifice and flows out from one or more layers of material dispensed from the dispensing orifice.

[0125] In some embodiments, the system includes components for removing excess fluid (e.g., excess sheath fluid and / or excess buffer solution) from a receiving surface, whereby the fiber structure dispensed from an orifice of the printhead may optionally be deposited from the surface of the dispensed fiber structure. During printing, excess fluid may collect or “pool” on the receiving surface or the surface of the dispensed fiber structure. Such pooling can interfere with the deposition process. For example, pooled sheath fluid may cause the dispensed fibers to slip from their intended location in the 3D structure being printed. Therefore, in some embodiments, removing excess sheath fluid from the receiving surface and optionally from the surface of the dispensed fiber structure via a fluid removal component can improve the additive manufacturing of three-dimensional structures.

[0126] Excess fluid can be removed from the receiving surface or from one or more layers of the distributed fibers by drawing fluid from those surfaces, by allowing or facilitating the evaporation of fluid from those surfaces, or, in embodiments where the receiving surface is porous, by drawing excess fluid through the porous surface. In some embodiments, the receiving surface comprises a porous material with pores sized to facilitate fluid passage and sized to support one or more layers of the fiber structure deposited thereon.

[0127] In some embodiments, components for removing excess fluid from the receiving surface and optionally from the surface of the dispensed fibrous structure may be included in a system configured to dispense material into a multi-walled plate or petri dish. In some embodiments, the receiving surface on the printing bed includes or is placed close to an absorbent material, which facilitates the absorption of excess fluid from the receiving surface. For example, a perforated plate insert having a bottom made of a porous membrane material or any other porous membrane substrate may be placed on top of or near an absorbent material such as a sponge. The absorbent material is used to draw excess fluid away 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 accumulating 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., on the periphery of the receiving surface so as not to interfere with the deposition of the dispensed material), excess sheath fluid can be drawn from the receiving surface into the absorbent material.

[0128] In some embodiments, the receiving surface includes one or more tubes fluidly coupled to a vacuum source, which can provide suction to remove excess fluid from the receiving surface and optionally from the surface of the dispensed fiber structure. In such embodiments, a solid or porous receiving surface may also be used. In some embodiments, the printhead is configured to further include one or more vacuum channels, each having an orifice located near (i.e., adjacent to) a dispensing orifice. Each of the one or more vacuum channels has an inlet configured to facilitate fluid communication with one or more vacuum channels. When the printhead is in fluid communication with the vacuum channels, the one or more vacuum channels direct negative pressure to a region of the receiving surface where material is being dispensed or has already been dispensed from the dispensing orifice and / or a portion of the surface region of the dispensed fiber structure, thereby drawing excess fluid from the receiving surface and optionally from the surface of the dispensed fiber structure, thus eliminating fluid pooling on the receiving surface and / or the dispensed fiber structure.

[0129] In some embodiments, one or more vacuum tubes are at least partially disposed in one or more extensions protruding from the printhead, the extensions protruding in the same generally direction as the extension including the dispensing orifice and dispensing channel 110. In such embodiments, the one or more extensions including the vacuum tubes do not extend further than the extension including the dispensing orifice and dispensing channel 110 so as not to interfere with the dispensing process.

[0130] In some embodiments, the fluid removal feature can be a characteristic of the fluid composition itself. For example, the sheath fluid composition and / or buffer solution composition can be designed to evaporate after dispensing from the dispensing orifice, thereby eliminating the accumulation of excess fluid on the receiving surface or the surface of the dispensing fibrous structure. For example, the sheath fluid may have a boiling point that causes evaporation after dispensing while remaining in a liquid state before dispensing.

[0131] In some embodiments, the printing system includes a 3D motorized stage comprising three arms for positioning a print head and dispensing orifices in three-dimensional space above a print bed, the print bed including a surface for receiving printing material. In one embodiment, the 3D motorized stage (i.e., the positioning unit) can be controlled to position a vertical arm extending along the z-axis of the 3D motorized stage such that the print head orifice points downwards. A first horizontal arm extending along the x-axis of the motorized stage is fixed to a fixed base platform. A second horizontal arm extending along the y-axis of the motorized stage is movably coupled to the upper surface of the first horizontal arm such 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.

[0132] In some embodiments, the receiving surface is located on top of a platform 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 a programmable controller processor, such as a computer. In a preferred embodiment, the print head and the receiving surface move together along all three principal axes of a Cartesian coordinate system via the 3D motorized stage, and the movement of the stage is defined using computer software. It should be understood that the invention is not limited to the described positioning system, and other positioning systems are known in the art. When material is dispensed from a dispensing orifice on the print head, the positioning unit moves in a software-controlled mode, thereby creating a first layer of dispensed material on the receiving surface. Additional layers of dispensed material are then stacked on top of another layer, such that the final 3D geometry of the dispensed material layers is typically a replica of a 3D geometry design provided by the software. The 3D design can be created using typical 3D CAD (Computer-Aided Design) software or generated from digital images, as known in the art. Furthermore, if the software-generated geometry contains information about the specific material to be used, specific input material types can be assigned to different geometric locations according to one embodiment of the invention. For example, in some implementations, the printed 3D structure may include two or more different input materials, each with different properties (e.g., each input material may include different cell types, different cell concentrations, different ECM compositions, etc.).

[0133] Aspects of the printing system of the present invention include software programs configured to facilitate the deposition of the input material of the present invention in a specific pattern and at specific locations to form specific fibers, planar, or 3D structures. To manufacture such structures, the printing system of the present invention deposits the input material of the present invention at precise locations (in two or three dimensions) on a receiving surface. In some embodiments, the location where the printing system deposits the material is defined by user input and translated into computer code. In some embodiments, the computer code includes a sequence of instructions executable in the central processing unit (CPU) of a digital processing device, written to perform a specified task. In some embodiments, printing parameters, including but not limited to, print fiber size, pump speed, printhead positioning system travel speed, 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.

[0134] Aspects of the present invention include a method 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 according to the commands to form a structure having a defined geometry.

[0135] In some embodiments, the location where the printing system deposits input material is defined by user input and translated into computer code. In some embodiments, the apparatuses, systems, and methods disclosed herein also include non-transitory computer-readable storage media or storage media encoded with computer-readable program code. In some embodiments, the computer-readable storage medium is a tangible component of a digital processing device, such as a bioprinter (or a component thereof) or a computer connected to a bioprinter (or a component thereof). In some embodiments, the computer-readable storage medium may optionally be removable from the digital processing device. In some embodiments, as non-limiting examples, the computer-readable storage medium includes CD-ROMs, DVDs, flash memory devices, solid-state storage, disk drives, magnetic tape drives, optical disc drives, cloud computing systems and / or services, etc. In some cases, programs and instructions are permanently, substantially permanently, semi-permanently, or non-transitory encoded on the storage medium.

[0136] In some embodiments, the apparatus, systems, and methods described herein include software, server, and database modules. In some embodiments, a "computer module" is a software component (including code segments) that interacts with a larger computer system. In some embodiments, a software module (or program module) appears as one or more files and typically handles specific tasks within a larger software system.

[0137] In some embodiments, the module is included in one or more software systems. In some embodiments, the module is integrated into one or more other modules within one or more software systems. A computer module may optionally be a standalone code segment, or may optionally be code that is not individually identifiable. In some embodiments, the module resides in a single application. In other embodiments, the module resides in multiple applications. In some embodiments, the module is hosted on one 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 embodiments of the present invention allows an end user to use a computer to perform one or more aspects of the methods described herein.

[0138] 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 application input and output, as well as hierarchical or other data structures for storing information. In some embodiments, the computer module includes a display screen. In other embodiments, the computer module presents a two-dimensional GUI via the display screen. In some embodiments, the computer module presents a three-dimensional GUI via the display screen, such as a virtual reality environment. In some embodiments, the display screen is a touchscreen and presents an interactive GUI.

[0139] Aspects of the invention include one or more quality control components configured to monitor and / or adjust one or more parameters of the printing system of the invention to ensure that one or more printed fibers have suitable characteristics. For example, in some embodiments, if the deposition process proceeds too quickly, a printed fiber structure may begin to form a coiled structure within or outside the distribution channel 110 after distribution has been performed. In some embodiments, the quality control components include a camera configured to monitor the deposition process by collecting one or more images of the printed fiber structure and to determine whether a coiled structure has formed. In some embodiments, the quality control components are configured to adjust one or more parameters of the deposition process (e.g., reducing pressure and / or reducing deposition rate) to reduce or avoid the formation of a coiled structure by the printed fiber structure.

[0140] Aspects of the invention include one or more fluid reservoirs configured to store fluid and deliver it to a printing system (e.g., a printhead) via one or more fluid channels, the fluid channels providing fluid communication between the printing system and the reservoir. In some embodiments, the printing system includes one or more fluid reservoirs in fluid communication with the fluid channels. In some embodiments, the fluid reservoir is connected to an inlet port of the fluid channel. In some embodiments, the fluid reservoir is configured to hold a fluid volume ranging from about 100 μL to about 1 L, 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.

[0141] In some embodiments, the printing system includes a pressure control unit fluidly coupled to one or more reservoirs. The pressure control unit is configured to provide force to move one or more fluids through the printing system. In some embodiments, the pressure control unit provides pneumatic pressure to one or more fluids via one or more connecting tubes. The applied pressure forces the fluid out of the reservoir and into the printhead through corresponding fluid channels. In some embodiments, alternative components may be used to move the fluid through the channels. For example, a series of electronically controlled injection pumps may be used to provide the force to move the fluid through the printhead.

[0142] In some embodiments, the printing system includes an optical module (as described above) for optionally exposing a photocrosslinkable input material to light to crosslink the material. The optical module according to embodiments of the invention may be integrated into the printhead or may be a component of the printing system.

[0143] Input materials:

[0144] Aspects of the invention include input materials that can be used to print fibrous structures. In some embodiments, the input material comprises a hydrogel. Non-limiting examples of hydrogels include alginate, agarose, collagen, fibrinogen, gelatin, chitosan, hyaluronic acid-based gels, or any combination thereof. A variety of synthetic hydrogels are known and can be used in embodiments of the systems and methods provided herein. For example, in some embodiments, one or more hydrogels form the structural basis of the printed three-dimensional structure. In some embodiments, the hydrogel has the ability to support the growth and / or proliferation of one or more cell types, which may be dispersed within the hydrogel or added to the hydrogel after it has been printed in a three-dimensional structure. In some embodiments, the hydrogel may be crosslinked by a chemical crosslinking agent. For example, hydrogels comprising alginate may be crosslinkable in the presence of divalent cations, hydrogels comprising chitosan may be crosslinkable using polyvalent anions such as sodium tripolyphosphate (STP), hydrogels comprising fibrinogen may be crosslinkable in the presence of enzymes such as thrombin, and hydrogels comprising collagen, gelatin, agarose, or chitosan may be crosslinkable in the presence of heat or alkaline solutions. In some embodiments, hydrogel fibers can be produced via a precipitation reaction, which is achieved through solvent extraction from the input material upon exposure to a crosslinking agent material miscible with the input material. Non-limiting examples of input materials for forming fibers via precipitation reactions include collagen and polylactic acid. Non-limiting examples of crosslinking materials capable of forming precipitation-mediated hydrogel fibers include polyethylene glycol (PEG) and alginate. Crosslinking of the hydrogel increases its stiffness, thereby allowing the formation of a cured hydrogel in some embodiments.

[0145] In some embodiments, the hydrogel comprises alginate. When contacted with a divalent cation, alginate forms a cured colloidal gel (high water content gel or hydrogel). Any suitable divalent cation can be used to form a cured hydrogel with an input material containing alginate. In the alginate ion affinity series, Cd... 2+ >Ba 2+ >Cu 2+ >Ca 2+ Ni 2+ >Co 2+ >Mn 2+ In, Ca 2+ The most characteristic and most commonly used method for forming 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 alginate, Section 3: Manufacturing and Structure of Alginate, *Alginate: Scientifically Controlled Aquatic Products*, 2000, International Specialty Products: Santiago, pp. 4–7). G-rich alginates tend to form thermally stable, strong, and brittle calcium gels, while M-rich alginates tend to form less thermally stable, weaker, but more elastic gels. In some embodiments, the hydrogel comprises depolymerized alginates as described in U.S. Provisional Patent Application No. 62 / 437,601, the disclosure of which is incorporated herein by reference in its entirety.

[0146] In some embodiments, the hydrogel can be crosslinked using a free radical polymerization reaction to create covalent bonds between molecules. Free radicals can be generated by exposing a photoinitiator to light (typically ultraviolet light), or by exposing the hydrogel precursor to a chemical source of free radicals, such as ammonium persulfate (APS) or potassium persulfate (KPS), in combination with N,N,N,N-tetramethylethylenediamine (TEMED) as both an initiator and a catalyst. Non-limiting examples of photocrosslinkable hydrogels include methacrylated hydrogels, such as gelatin methacrylate (GEL-MA), or polyethylene glycol diacrylate (PEG-DA)-based hydrogels, which are used in cell biology due to their ability to crosslink in the presence of free radicals after exposure to ultraviolet light and because of their inertness to cells. PEG-DA is commonly used as a scaffold in tissue engineering because polymerization occurs rapidly at room temperature and requires low energy input, it has high water content, is flexible, and can be tailored to contain a variety of biomolecules.

[0147] Additional components:

[0148] The input material according to embodiments of the invention may include any of a variety of natural or synthetic polymers that support the viability of living cells, including, for example, laminin, fibroin, hyaluronic acid, polyethylene glycol-based gels, gelatin, chitosan, agarose, or combinations thereof. In a particularly preferred embodiment, the bio-ink composition of the invention is physiologically compatible, i.e., conducive to cell growth, differentiation, and communication. In some embodiments, the input material includes one or more physiological matrix materials or combinations thereof. “Physiological matrix material” refers to a biomaterial found in natural mammalian tissues. Non-limiting examples of such physiological matrix materials include: fibronectin, platelet-reactive proteins, glycosaminoglycans (GAGs) (e.g., hyaluronic acid, chondroitin 6-sulfate, dermatan sulfate, chondroitin 4-sulfate, or keratin sulfate), deoxyribonucleic acid (DNA), adhesion glycoproteins, and collagen (e.g., collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, or collagen XVIII).

[0149] Collagen provides tensile strength to most tissues, and multiple collagen fibrils, each approximately 100 nm in diameter, bind together to form strong, coiled helical fibers approximately 10 μm in diameter. The biomechanical functions of certain tissue structures are conferred through the directional alignment of collagen fibers. In some embodiments, the input material includes collagen fibrils. Input materials including collagen fibrils can be used to generate fibrous structures that form tissue structures. By adjusting the diameter of the fibrous structure, the orientation of the collagen fibrils can be controlled, thereby guiding the polymerization of the collagen fibrils in the desired manner.

[0150] For example, previous studies have shown that microfluidic channels of varying diameters can guide collagen fibrils to polymerize and form fibers oriented along the channel length, but this is limited to channels with diameters of 100 μm or smaller (Lee et al., 2006). Primary endothelial cells grown in these oriented matrices show alignment along the direction of the collagen fibers. In another study, Martinez et al. demonstrated that 500 μm channels within beaded cellulose scaffolds can guide collagen and cell alignment (Martinez et al., 2012). In some embodiments, the input material can be formed into a fibrous structure with a diameter ranging from about 20 μm to about 500 μm, for example, about 50 μm, about 75 μm, about 100 μm, about 125 μm, about 150 μm, about 175 μm, about 200 μm, about 225 μm, about 250 μm, about 275 μm, about 300 μm, about 325 μm, about 350 μm, about 375 μm, about 400 μm, about 425 μm, about 450 μm, or about 475 μm. By adjusting the fiber diameter, the orientation of collagen fibers within the fibrous structure can be controlled. Therefore, the fibrous structure and the collagen fibers within it can be patterned to produce a tissue structure with a desired collagen fiber arrangement, which is crucial for imparting the desired biomechanical properties to the 3D printed structure.

[0151] Mammalian cell types:

[0152] The input material according to embodiments of the present invention may be a combination of 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 heart cells), and ectoderm cells (skin, nervous system or eye cells), or any combination thereof.

[0153] In some implementations, the input materials may include: fibroblasts, chondrocytes, meniscal 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, chondrocytes, osteoblasts, osteoclasts, and any combination thereof.

[0154] Cells can be obtained from a donor (exogenous) or a recipient (autologous). Cells can also originate from established cell culture lines, or they can be genetically engineered and / or manipulated to achieve a desired genotype or phenotype. In some embodiments, tissue slices may also be used, which can provide multiple different cell types within the same structure.

[0155] In some embodiments, the cells may be obtained from a suitable donor (human or animal) or from the subject to whom the cells will be implanted. Mammal species include, but are not limited to, humans, monkeys, dogs, cattle, horses, pigs, sheep, goats, cats, mice, rabbits, and rats. In one embodiment, the cells are human cells. In other embodiments, the cells may be derived from animals, such as dogs, cats, horses, monkeys, or any other mammal.

[0156] Appropriate growth conditions for mammalian cells are well known in the art (Freshney, RI (2000), Animal Cell Culture, Basic Techniques Handbook; Hoboken, NJ; John Wiley & Sons; 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 supplementary elements such as growth factors, salts, minerals, vitamins, etc., which can be selected depending 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 Durbeco Modified Eagle 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, which is as suitable as various other standard media known to those skilled in the art. Growth conditions will vary depending on the type of mammalian cells used and the tissue required.

[0157] In some implementations, cell type-specific agents can be advantageously used in subject input materials for the corresponding cell type. For example, extracellular matrix (“ECM”) can be extracted directly from the tissue of interest, 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 derived from, for example, zPredicta (rBone) TM (Available at zpredicta.com / home / products) can be purchased from the supplier.

[0158] Surfactant:

[0159] 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 growth factor, pleiotropic growth factor, endothelin, nicotinamide, glucagon-like peptide-1, glucagon-like peptide-II, parathyroid hormone, tendinin-C, elastin, thrombin-derived peptide, laminin, bioactive peptides containing cell-binding domains and heparin-binding domains, therapeutic agents, and any combination thereof.

[0160] As used herein, the term "therapeutic agent" means any chemical component of a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject. Non-limiting examples of therapeutic agents (also referred to as "medicines") are described in well-known references such as the Merck Index, the Physician's Desk Reference, and the Pharmacological Basis of Therapeutics, and these include, but are not limited to, drugs; vitamins; mineral supplements; substances intended to treat, prevent, diagnose, cure, or alleviate a disease or ailment; substances affecting the structure or function of the body; or prodrugs that, upon exposure to a physiological environment, become biologically active or more active. In some embodiments, one or more therapeutic agents may be used that are capable of being released from the input material described herein into adjacent tissues or fluids after implantation into a subject. Examples of therapeutic agents include, but are not limited to, antibiotics, anesthetics, any therapeutic agent that promotes regeneration or tissue healing or relieves pain, infection, or inflammation, or any combination thereof.

[0161] Additional active agents may include, but are not limited to, proteins, peptides, nucleic acid analogs, nucleotides, oligonucleotides, nucleic acids (DNA, RNA, siRNA), peptide nucleic acids, aptamers, antibodies or fragments or portions thereof, antigens or epitopes, hormones, hormone antagonists, growth factors or recombinant growth factors and their fragments and variants, cytokines, enzymes, antibiotics or antimicrobial compounds, anti-inflammatory agents, antifungal agents, antiviral agents, toxins, prodrugs, small molecules, drugs (e.g., drugs, dyes, amino acids, vitamins, antioxidants), or any combination thereof.

[0162] Non-limiting examples of antibiotics suitable for inclusion in input materials include: aminoglycosides (e.g., neomycin), salinomycins, carbapenems, carbapenems, cephalosporins (e.g., cefazolin, cefaclor, ceftoran, cefotaxime), glycopeptides (e.g., vancomycin), macrolides (e.g., erythromycin, azithromycin), monocyclic cyclophosphamides, penicillins (e.g., amoxicillin, ampicillin, cloxacillin, dicloxacillin, flucloxacillin), polypeptides (e.g., bacitracin, polymyxin B), and quinolones. Nortocin (e.g., ciprofloxacin, enoxacin, gatifloxacin, ofloxacin, etc.), sulfonamides (e.g., sulfasalazine, trimethoprim, trimethoprim-sulfamethoxazole (compound sulfamethoxazole)), tetracyclines (e.g., doxycycline, minocycline, tetracycline, etc.), chloramphenicol, lincomycin, clindamycin, ethambutol, mupirocin, metronidazole, pyrazinamide, thiamphenicol, rifampin, thiamphenicol, dapsone, clofazimine, quinupristin, metronidazole, linezolid, isoniazid, fosfomycin, fusidic acid, or any combination thereof.

[0163] Non-limiting examples of antibodies include: abciximab, adalimumab, alemtuzumab, basilizumab, bevacizumab, cetuximab, cetozumab, dacrolimus, eculizumab, efazolinumab, gemtuzumab, teimozumab, infliximab, moromona-CD3, natezumab, ofamumab, omalizumab, palizumab, panitumumab, ranibizumab, rituximab, tosimomab, trastuzumab, pentiazem, asimozumab, tosimomab, betomozumab, belimumab, besoxumab, bismuth subcitrate. Anti-, canatumab, calocumab pendibutide, caputuzumab, denosumab, ezolizumab, ifencoumarab, ertosumab, dazumab, fasomumab, fentozumab, gemtuzumab, incemab, labezumab, mepolizumab, morivuzumab, nimotuzumab, nofetomumab, ozovumab, petumumab, pertuzumab, lovezumab, rulizumab, thioxolumab, tetazumab, tefezumab, tocilizumab, uterotumab, vexizumab, votomumab, zalumab, zalumab, or any combination thereof.

[0164] Non-limiting examples of enzymes suitable for use with the input materials described herein include: peroxidase, lipase, amylose, organophosphate dehydrogenase, ligase, restriction endonuclease, ribonuclease, DNA polymerase, glucose oxidase, and laccase.

[0165] Additional non-limiting examples of active agents suitable for use with the input materials of this invention include: cell growth media, such as Durbeco modified Eagle 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 morphogenetic growth factor, bone morphogenetic-like proteins, 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 (RGLandes 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 combinations of analgesics, anti-inflammatory agents, steroids, or any combination thereof.

[0166] Additional fluid:

[0167] Aspects of the invention include one or more buffer solutions. Buffer solutions according to embodiments of the invention are miscible with the input material (e.g., hydrogel) and do not crosslink the input material. In some embodiments, the buffer solution comprises an aqueous solvent. Non-limiting examples of buffer solutions include polyvinyl alcohol, water, glycerol, propylene glycol, sucrose, gelatin, or any combination thereof.

[0168] The viscosity of the buffer solution according to embodiments of the 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 buffer solution can be adjusted to match the viscosity of one or more input materials.

[0169] Aspects of the invention include one or more sheath fluids. A sheath fluid according to an embodiment of the invention is a fluid that can be used at least partially to encapsulate or “wrap” input material dispensed from dispensing channel 110. In some embodiments, the sheath fluid comprises an aqueous solvent. Non-limiting examples of sheath fluids include polyvinyl alcohol, water, glycerol, propylene glycol, sucrose, gelatin, or any combination thereof. The viscosity of the sheath fluid according to embodiments of the 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.

[0170] In some embodiments, the sheath fluid includes a chemical crosslinking agent. In some embodiments, the chemical crosslinking agent comprises 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 is used. 2+ As a divalent cation. In some embodiments, the concentration of the divalent cation in the sheath fluid ranges from about 80 mM to about 140 mM, for example about 90, 100, 110, 120 or 130 mM.

[0171] How to use:

[0172] The present invention includes methods for printing linear fiber structures, planar structures comprising one or more fiber structures, or three-dimensional (3D) structures comprising two or more layers of planar structures. In some embodiments, a method first includes providing a design for the planar or 3D structure to be printed. The design can be created using commercially available CAD software. In some embodiments, the design includes information about specific materials (e.g., for heterogeneous structures comprising multiple materials), which will be assigned to specific locations within the structure to be printed.

[0173] In some implementations, a method includes using a 3D printer comprising: a printhead; a receiving surface for receiving material dispensed by the printhead; and a positioning unit operatively coupled to the receiving surface for positioning the printhead in a 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.

[0174] Aspects of the method include providing one or more input materials to be dispensed by a printhead. In some embodiments, one or more cell types are compatible with the input material and optionally dispensed within the input material. In some embodiments, a sheath fluid is used as a lubricant to lubricate movement of the input material within the printhead. In some embodiments, the sheath fluid includes a crosslinking agent for curing at least a portion of the hydrogel before or during dispensing from the printhead.

[0175] Aspects of the method include transmitting a design to a 3D printer. In some embodiments, communication may be implemented, for example, via a programmable control processor. In some embodiments, the method includes controlling the relative positioning of the print head and the receiving surface in three-dimensional space, and simultaneously dispensing sheath fluid and input material from the print head, individually or in combination. In some embodiments, the material dispensed from the print head is dispensed coaxially, such that the sheath fluid encapsulates the input material. This coaxial arrangement allows a crosslinking agent in the sheath fluid to solidify the input material, thereby producing a solidified fibrous structure dispensed from the print head.

[0176] In some embodiments, a method includes: depositing a first layer of a distributed fiber structure onto a receiving surface, the first layer comprising an arrangement of fiber structures specified by design, and iteratively repeating the deposition steps; depositing subsequent fiber structures onto the first and subsequent layers, thereby depositing the distributed fiber structures layer by layer in a design-specified geometric arrangement to produce a 3D structure.

[0177] In some implementations, multiple input materials, such as multiple hydrogels, are deposited in a controlled sequence, at least some of which include one or more cell types, thereby allowing a controlled arrangement of input materials and cell types to be deposited in a design-specified geometric arrangement.

[0178] In some embodiments, a method includes removing excess fluid from a receiving surface and optionally from the surface of the dispensed fiber structure. For example, the excess fluid removal step can be performed continuously throughout the printing process to remove excess fluid that would otherwise interfere with the layering of the dispensed fiber structure in a geometry provided by the design. Alternatively, the excess fluid removal step can be performed intermittently or simultaneously with one or more deposition steps throughout the printing process. In some embodiments, excess fluid removal is achieved by extracting fluid from the receiving surface and optionally from the surface of the dispensed fiber structure. In some embodiments, excess fluid removal is achieved by aspirating excess fluid through a receiving surface, the receiving surface including orifices sized to allow fluid passage. In some embodiments, excess fluid removal is achieved by providing fluid that has evaporated after being dispensed from the dispensing orifice.

[0179] Aspects of the present invention include a method for manufacturing a 3D structure comprising one or more input materials. The 3D structure can be used to repair and / or replace at least a portion of damaged or diseased tissue in a subject.

[0180] As described above, any suitable divalent cation can be used in conjunction with the method of the present invention to cure chemically crosslinkable 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 is used. 2+ As a divalent cation. In a preferred embodiment, the chemically crosslinkable input material contains Ca. 2+ The solution contacts the solidified fibrous structure. In some embodiments, the Ca in the sheath fluid... 2+ The concentration ranges from about 80 mM to about 140 mM, for example about 90, 100, 110, 120 or 130 mM.

[0181] In some implementations, the input material cures in less than about 5 seconds, such as less than about 4 seconds, less than about 3 seconds, less than about 2 seconds, or less than about 1 second.

[0182] Aspects of the present invention include a method for depositing one or more input materials in a patterned manner using software tools to form a solidified structural layer, said solidified structural layer being formed as a multilayer 3D tissue structure. In some embodiments, the multilayer 3D tissue structure comprises multiple mammalian cells. Advantageously, by adjusting the composition of the input materials of the present invention (e.g., mammalian cell type, cell density, matrix composition, surfactant), the method of the present invention can be used to create multilayer 3D tissue structures, wherein the multilayer 3D tissue structure has a precisely controlled composition at any specific location in three-dimensional space. Therefore, the method of the present invention facilitates the production of complex three-dimensional tissue structures.

[0183] In some embodiments, the method includes simultaneously dispensing a buffer solution and / or sheath fluid through a core channel, one or more input materials through one or more shell channels, and sheath fluid through a sheath flow channel 118 to form a hollow core in the printed fiber.

[0184] 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 crosslinking agent, and contact occurs at the sheath fluid crossover point to solidify the outer surface of the crosslinkable material flow in the distribution channel 110.

[0185] In some embodiments, the non-crosslinkable material in the core channel comprises a chemical crosslinking agent 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.

[0186] In some embodiments, the non-crosslinkable material in the core channel includes a chemical crosslinking agent, and the sheath fluid in the sheath flow channel 118 includes a chemical crosslinking agent, 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.

[0187] In some embodiments, the system includes a printhead that includes a core channel that includes at least two core inlet sub-channels connected to different fluid reservoirs that include different input materials, and the method includes alternately dispensing different input materials through shell inlet sub-channels 126 to produce a cured fiber structure comprising different core materials along the length of a continuous fiber.

[0188] In some embodiments, the system includes a printhead that includes a first shell channel that includes at least two shell inlet sub-channels 126 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 a continuous fiber.

[0189] In some embodiments, the system includes a printhead having at least two shell channels connected to different fluid reservoirs containing different input materials, and the method includes simultaneously dispensing different input materials through the first and second shell channels to produce a cured fiber structure comprising different concentric shells.

[0190] In some embodiments, the system includes a printhead comprising a first shell channel including at least two shell inlet sub-channels 126 connected to different fluid reservoirs comprising reinforcing hydrogel material and biocompatible hydrogel material; and the method includes alternately dispensing reinforcing hydrogel material and biocompatible hydrogel material through dispensing channels 110 to generate perfusionable tissue fibers. In another embodiment, the printhead further includes a second shell channel 128, and the method further includes dispensing the same or different reinforcing hydrogel material through the second shell channel 128 to generate a concentric second shell surrounding the first shell.

[0191] In an alternative implementation, solid fibers can be produced as a means of printing other non-printable materials. This method allows selection of easily printable shell materials, such as alginate, and core materials that would otherwise be unprintable, such as pure collagen. Here, the ability to switch core materials allows the user to further control the core composition. The core material can contain different cell types and be arranged or combined along the fiber length. In this case, the shell material can also be switched and can also contain different cell types.

[0192] Practicality:

[0193] In some embodiments, structures produced using the systems and methods provided herein can be used in the field of drug discovery, where, for example, determining cellular responses to various compounds and compositions is meaningful. The use of planar and 3D cell cultures fabricated using embodiments of the systems and methods provided herein can provide experimental conditions that more closely resemble in vivo cellular and tissue conditions compared to conventional 2D cell cultures. The 3D arrangement of cells can more closely mimic in vivo cell-cell interactions and responses to external stimuli, and the heterogeneity of the 3D structures produced using the systems and methods provided herein allows for the study of tissues and potential organs. It is anticipated that structures containing 3D cells fabricated using embodiments of the systems and methods provided herein can provide similar benefits to the cosmetics industry by offering an alternative means of testing cosmetics.

[0194] In some implementations, aspects of the systems and methods provided herein are compatible with standard orifice plate technology. In the methods and systems provided herein, orifice plates or orifice plate inserts can be used with or as part of a print bed. Therefore, the various implementations of the systems and methods provided herein are compatible with instrumentation and practices utilizing orifice plates, thereby allowing them to be easily integrated into existing process flows.

[0195] In some embodiments, one or more fluid channels within the printhead of the present invention are compatible with other microfluidic modules. For example, known microfluidic modules may be included in the printhead of a system provided herein upstream of the dispensing orifice. Such modules may include, for example, cell counting, cell sorting, cell analysis, and / or concentration gradient generation modules.

[0196] In some implementations, the throughput of 3D printing can be increased by adding additional printheads to the system in parallel. Each printhead contains all the components needed to print multi-material structures, so multiple 3D structures can be printed simultaneously by including additional printheads in the system.

[0197] All patents and patent applications cited in this article are hereby incorporated in their entirety by reference.

[0198] Although the invention has been described in detail by way of illustration and examples for purposes of clarity, it will be readily apparent to those skilled in the art, in view of the teachings of the invention, that certain changes and modifications may be made without departing from the spirit or scope of the appended claims.

[0199] This invention enables multi-material switching, thus allowing modification of the vessel wall composition (cell type and biomaterial composition) along the length of the channel during continuous printing. This example illustrates how, to reproduce the biological structure and function of a renal tubule, the wall composition at the proximal end will differ from that at the distal end. Alternatively, in perfusionable printed 3D liver tissue, the vessel wall may be lined with low-permeability portal venous endothelial cells at the larger opening end of the vessel, and with higher-permeability sinusoidal endothelial cells in vessels with narrower channels to model the sinusoidal space. Similar to liver tissue, it is necessary to investigate the interaction between the contents of the perfusion channel and different stromal cell types outside the channel in one or more shells. This can be applied to generate multi-tissue toxicity models incorporating shear flow effects. Individual tissues can be printed where the cellular contents of the fibrous shell are switched along its length to produce encoded hollow fibers with different regions corresponding to different organ types. Such shell content switching is not possible with systems based on non-microfluidic injectors.

[0200] Example:

[0201] Example 1: Perfusionable tissue fibers

[0202] Bioprinted perfusionable tissue fibers with a liquid core and a cell-containing gel shell have significant commercial and clinical implications, allowing needles to be inserted into the fiber core and attached to a pump after printing, which actuates the fluid of interest through the fiber. In this way, one can simulate the flow of nutrients, drugs, or other compounds of interest through cell-containing fibers. However, a significant challenge arises when attaching the needle to the fiber, as the mechanical requirements for such attachment differ greatly from those required to support functional biology. Therefore, the ability to switch the shell material in real time during printing allows users to use a robust material for printing in areas intended for attaching the needle (fiber tip) and a material containing soft cells for printing in areas intended to support biological functions.

[0203] like Figure 12 As shown, the perfusionable tissue fibers according to the invention are bioprinted using a reinforced hydrogel material (blue) consisting of 4 wt% low-viscosity sodium alginate, a biocompatible hydrogel material (red) consisting of 1.3 wt% of the same alginate, and a core consisting of 3% polyvinyl alcohol. The fibers are then attached using standard 30-gauge stainless steel Luer lock needles, and a mixture of gelatin and transglutaminase is used to seal the needle / fiber connection to prevent leakage during perfusion.

[0204] Furthermore, even with appropriate reinforcement at the ends of the infusible fibers to achieve proper connections, breakage still occurs with increasing fiber length, including softer, cellular material, particularly at higher flow rates. Therefore, as... Figure 13As shown, the invention further considers optionally adding a second concentric shell comprising the same or different reinforcing hydrogel material, such that the fiber is supported along its entire length.

[0205] The foregoing description merely illustrates the principles of the invention. It should be understood that those skilled in the art will be able to design various arrangements that, while not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all embodiments and conditional language described herein are primarily intended to aid the reader in understanding the principles of the invention and the conception of the facilitating techniques provided by the inventors, and should be interpreted as not constituting a limitation on such specifically described embodiments and conditions. Moreover, all statements herein recounting the principles and aspects of the invention, as well as specific embodiments thereof, are intended to cover both structural and functional equivalents. Additionally, it is intended that such equivalents include both currently known equivalents and future-developed equivalents, i.e., any element developed to perform the same function regardless of its structure. Therefore, the scope of the invention is not intended to be limited to the exemplary aspects shown and described herein. Rather, the scope and spirit of the invention are embodied in the appended claims.

Claims

1. A printhead for a 3D printer, the printhead comprising a plurality of stacked layers forming a plurality of fluid channels, the printhead comprising: A core channel having at least one inlet, an outlet, and one or more fluid switches; A first shell channel, the first shell channel having at least one inlet, an outlet and one or more fluid switches; Multi-channel housing; as well as Channel allocation The multi-channel housing includes the core channel outlet, the first housing channel outlet, and the first fluid focusing chamber. The core channel outlet is located in the central region of the multi-channel housing and is in fluid communication with the inlet of the first fluid focusing chamber. The core channel outlet extends to a first vertical depth within the multi-channel housing, preferably wherein the core channel outlet extends to the first vertical depth within the first fluid focusing chamber, aligned with the distribution channel. The first shell channel outlet is concentrically disposed around the core channel and is in fluid communication with the inlet of the first fluid focusing chamber. The first shell channel outlet extends to a second vertical depth within the multi-channel shell, preferably / optionally wherein the first shell channel outlet extends to the first vertical depth within the first fluid focusing chamber, and The first fluid focusing chamber converges toward the distribution channel, and preferably the first fluid focusing chamber includes a truncated conical shape configured to focus fluid toward the distribution channel.

2. The printhead according to claim 1, wherein the first shell channel outlet has a gradient width, the gradient width increasing with the depth of entry into the multi-channel shell.

3. The printhead according to 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 printhead according to claim 1, further comprising: The second shell channel has at least one inlet and one outlet, wherein the outlet of the second shell channel is concentrically disposed around the outlet of the first shell channel in the multi-channel shell in the 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 housing channel outlet extends further into the multi-channel housing and / or the first fluid focusing chamber than the second housing channel outlet.

6. The printhead according to claim 1, further comprising: The second shell channel has at least one inlet and one outlet, and A second multi-channel housing is located between the first fluid focusing chamber and the distal end of the distribution channel. The second multi-channel housing includes the distribution channel, the second housing channel outlet, and the second fluid focusing chamber. The distribution channel is located in the 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 within the multi-channel housing. Preferably, the outlet of the distribution channel extends to the first vertical depth within the fluid focusing chamber. The second shell channel outlet is concentrically disposed around the distribution channel and is in fluid communication with the inlet of the second fluid focusing chamber, extending to a second vertical depth within the multi-channel shell. The second fluid focusing chamber converges toward the distribution channel, and preferably the second fluid focusing chamber includes a truncated conical shape configured to focus fluid toward the distribution channel.

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

8. The printhead according to any one of claims 1 to 7, wherein the first shell channel and / or the second shell channel further comprises at least one fluid distribution orifice configured to circumferentially distribute fluid around the outlet of the first shell channel and / or the outlet of the second shell channel.

9. The printhead of claim 8, wherein the at least one fluid distribution orifice connects the inlet of the first and / or second shell channel to the vertex of the upper curved surface of the outlet of the first and / or second shell channel; preferably wherein the upper curved surface has a parabolic or elliptical shape.

10. The printhead of claim 8 or 9, wherein the first and / or second shell channel inlet comprises two or more sub-channels configured to deliver fluid to the same or separate fluid distribution orifices.

11. The printhead of claim 10, wherein each sub-channel includes a fluid distribution orifice connecting the inlet of the first and / or second shell channel to the vertex of the upper curved surface of the corresponding first and / or second shell channel outlet, preferably wherein the upper curved surface has a parabolic or elliptical shape.

12. The printhead of claim 10, wherein each sub-channel is configured to dispense a different material, preferably wherein the first and / or second shell channel comprises two fluid switches and each sub-channel is fluidly connected to a different fluid switch.

13. The printhead according to any one of the preceding claims further includes a sheath flow channel, the sheath flow channel converging with the dispensing channel at a sheath fluid chamber located at the distal end of the fluid focusing chamber and the dispensing channel; preferably wherein the sheath fluid chamber comprises a truncated conical shape configured to focus fluid toward the dispensing channel.

14. The printhead of claim 13, wherein the sheath flow channel comprises a plurality of sheath flow sub-channels that converge toward the distribution channel via a sheath flow chamber.

15. The printhead according to any one of claims 1 to 14, wherein the printhead includes at least two core inlet sub-channels that converge at or near the core channel outlet, the multi-channel housing, and / or the fluid distribution orifice; preferably wherein the at least two core inlet sub-channels converge in an adjacent preceding layer, or in the same layer of the printhead as the core channel outlet, the multi-channel housing, and / or the fluid distribution orifice.

16. The printhead of claim 15, wherein the core channel further comprises at least one fluid distribution orifice configured to circumferentially distribute fluid around the core channel outlet; preferably wherein the at least one fluid distribution orifice connects the apex of the converging core channel inlet to the upper curved surface of the core channel outlet; more preferably wherein the upper curved surface has a parabolic or elliptical shape.

17. The printhead of claim 15, wherein each core inlet subchannel is configured to dispense a different material, preferably wherein the core channel comprises two fluid switches, and each core inlet subchannel is fluidly connected to a different fluid switch.

18. 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 Fluid focusing chamber, the fluid focusing chamber converging towards the distribution channel, The core channel is in fluid communication with the fluid focusing chamber. The core channel extends longitudinally through the central region of the fluid focusing chamber and is aligned with the dispensing channel. The plurality of shell channels are concentrically arranged around the core channel in the same layer of the printhead and are in fluid communication with the fluid focusing chamber. The inner shell channel extends longer than the outer shell channel into the fluid focusing chamber. The core channel extends longer than any shell channel into the fluid focusing chamber, 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.

19. The printhead according to claim 18, further comprising: Multiple fluid distribution orifices are configured to circumferentially distribute fluid around multiple shell channels, wherein each of the multiple fluid distribution orifices individually connects a corresponding shell channel inlet to the apex of the upper curved surface of a corresponding shell channel outlet in the multiple shell channels.

20. The printhead of claim 18, wherein at least one of the plurality of shell channels has a gradient width that increases with increasing longitudinal depth into the shell.

21. A printhead comprising a plurality of stacked layers forming a plurality of fluid channels, the printhead comprising: The core channel includes at least two core inlet sub-channels, each having a different fluid reservoir, inlet orifice, and control valve, which converge to form a single core channel outlet in fluid communication with the first fluid focusing chamber, preferably wherein the at least two core inlet sub-channels converge at or near the core channel outlet; A first shell channel, comprising at least two shell inlet sub-channels, the shell inlet sub-channels having different fluid reservoirs, inlet orifices and control valves, converging to form a single shell channel outlet in fluid communication with a second fluid focusing chamber; optionally, wherein the first shell channel comprises three shell inlet sub-channels, one of which is connected to a fluid reservoir comprising a buffer solution; Channel allocation; The fluid focusing chamber converges toward the distribution channel, and preferably the fluid focusing chamber comprises a truncated conical shape configured to focus the fluid toward the distribution channel; as well as A sheath flow channel that converges with the distribution channel at a sheath fluid intersection located between the second fluid focusing intersection and the distal end of the distribution channel.

22. The printhead of claim 21, wherein the core channel further comprises at least one fluid distribution orifice configured to circumferentially distribute fluid around the core channel outlet; preferably wherein the at least one fluid distribution orifice connects the apex of the converging core channel inlet to the upper curved surface of the core channel outlet; more preferably wherein the upper curved surface has a parabolic or elliptical shape.

23. A system for producing fiber structures, the system comprising: The printhead according to any one of claims 1 to 22; as well as A positioning component for positioning the dispensing orifice of the printhead in three-dimensional space, wherein the positioning component is operatively coupled to the printhead.

24. The system of claim 23, further comprising a programmable control processor for controlling the positioning component and for controlling the flow rate of one or more fluids through the printhead.

25. The system of claim 23, further comprising a fluid removal assembly configured to remove excess fluid dispensed from the printhead, wherein the fluid removal assembly includes a porous membrane configured to allow the excess fluid to pass through, and / or wherein the fluid removal assembly includes a vacuum configured to aspirate the excess fluid.

26. The system of claim 23, further comprising a pressure control component configured to regulate the flow rate of the one or more fluids through the printhead.

27. A method for producing a core-shell fiber structure, the method comprising: A system for producing fiber structures is provided, the system comprising: The printhead according to any one of claims 1 to 22, The printhead is configured to dispense a variety of input materials through the core channel and the shell channel, wherein at least one of the input materials includes a crosslinkable material and a sheath solution through the sheath flow channel; A receiving surface for receiving the first layer of material dispensed from the printhead; A positioning component for positioning the dispensing orifice of the printhead in 3D space, wherein the positioning component is operatively coupled to the printhead; A programmable control processor for controlling the positioning component and for controlling the flow rate of one or more fluids through the printhead; A fluid reservoir comprising the plurality of input materials and a sheath solution, wherein the fluid reservoir is in fluid communication with the printhead; The crosslinkable material is brought into contact with the sheath solution in the dispensing channel to create a cured fiber structure; and The cured fiber structure is dispensed from the dispensing orifice of the printhead.

28. The method of claim 27, wherein the system includes a core channel comprising at least two core inlet sub-channels connected to different fluid reservoirs comprising first and second input materials, and the method includes dispensing the first and second input materials [alternatingly] through the shell inlet sub-channels to produce a cured fiber structure comprising different core materials along the length of a continuous fiber.

29. The method of claim 28, wherein the system further comprises a first shell channel including at least two shell inlet sub-channels respectively connected to different fluid reservoirs including a third and a fourth input material, and the method includes dispensing the third and fourth input materials [alternatingly] through the shell inlet sub-channels to produce a cured fiber structure including different shell materials along the length of the continuous fiber.

30. The method of claim 28, wherein the system further comprises at least two shell channels connected to different fluid reservoirs comprising a third and a fourth input material, 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.

31. The method according to any one of claims 27 to 30, further comprising: The programmable control processor is encoded using the planar structure to be printed; and The first layer of the cured fiber structure is deposited on the receiving surface to print the planar structure.

32. The method according to any one of claims 27 to 30, further comprising: The programmable control processor is encoded using the 3D structure to be printed; and Subsequent layers of the cured fiber structure are deposited on top of the planar structure to print a 3D structure.

33. A bioprinted tissue fiber manufactured by the method according to any one of claims 28 to 30, said bioprinted tissue fiber having a variable core and shell material over the entire length of the fiber.

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