Systems and methods for manufacturing bioprinted fiber structures

Abstract

Aspects of the present disclosure include a manufacturing platform that includes a frame having a plurality of struts for securing crosslinkable fibers during printing to support a bioprinted fiber structure during printing, patterning, and / or processing. The continuous length of crosslinkable fibers is printed around the plurality of struts during a 3D bioprinting process. The manufacturing platform enables the crosslinkable fibers to be suspended during one or more of printing, patterning, and / or processing. In this way, the bioprinted fiber structure includes a uniform outer surface and can be easily modified and / or further processed after printing and patterning are complete.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 342,118, filed May 15, 2022, the content of which is incorporated herein by reference in its entirety.

[0002] Field of Disclosure The present disclosure generally relates to three - dimensional (3D) printing and generating three - dimensional biological structures from digital files. Specifically, the present invention relates to systems and methods for generating cross - linkable fiber structures having a uniform outer surface during printing and patterning and facilitating post - print modification thereof.

Background Art

[0003] Tissue engineering techniques have long sought to create viable synthetic structures that can mimic target tissues and target organs using numerous materials and methods. Unfortunately, however, the practical realization of these synthetic structures still faces several important challenges. A suitable synthetic device must protect encapsulated cells and / or tissue fragments from the host immune system without impeding the passage of nutrients, oxygen, and secreted products (e.g., insulin). Further, the materials used in creating the synthetic structure must be biocompatible and have sufficient strength and elasticity to survive in vivo over a long period without causing harmful immunological reactions.

[0004] In particular, implantation into the body can induce a biological response controlled by both the innate and adaptive immune systems of the device, which is intended to neutralize the device, called the foreign body reaction (FBR). The cellular response to pathogens recognized as being too large to be phagocytosed is partially mediated by macrophages that overexpress extracellular matrix (ECM) proteins such as fibronectin, promote fibroblast-mediated fibrosis, and as a result, also produce fibrogenic factors that form a fibrous capsule around the device. This fibrous capsule can interfere with the function of the device, particularly when it contains a therapeutic cell population that needs access to the flow of nutrients and oxygen to perform their intended function.

[0005] A wide range of materials, from naturally occurring polymers to synthetic materials, have been described as generating a fibrotic response (Ward WK, J Diabetes Sci Technol. (2008); 2:768 - 777, Zhang L et al., Nature Biotech. (2013); 31:553 - 556, Ratner BD, Journal of Controlled Release. (2002), 78:211 - 218). Furthermore, physical parameters such as the shape, size, stiffness, and texture of synthetic tissue structures are inherent properties known to contribute to FBR. For example, the surface of synthetic tissue structures can influence the behavior of macrophages and other immune cells, and structures lacking sharp edges and having a smooth surface are generally highly biocompatible and induce less inflammation (Mariani E et al., Int J Mol Sci. (2019); 20:doi:10.3390 / ijms20030636, Salthouse TN, Journal of Biomedical Materials Research Part A. (1984); 18:395 - 401). Additionally, changes in surface roughness at the nanoscale are also associated with increased protein adsorption (Hulder M et al., Int J Nanomedicine. (2011); 6:2653 - 2666, Roach P., J Mater Sci Mater Med. (2007), 18:1263 - 1277, Scopellit PE et al., PLOS ONE. (2010), 5:e11862, Rechendorff K et al., Langmuir. (2006), 22:10885 - 10888, Hovgaard MB et al., J Phys Chem.B (2008); 112:8241 - 8249), and different nanostructured topographies can potentially affect cell interactions (Baker DW et al., Biomacromolecules. (2011); 12:997 - 1005, Jahed Z., Biomaterials. (2014); 35:9363 - 9371).

[0006] Accordingly, improvements in both design and materials are still needed to adapt to the opposing purposes of immune defense and nutrient passage and to assist in reducing the FBR response. Thus, there is a need for a synthetic tissue structure that effectively balances the ability to reduce or avoid recognition by the immune system and / or damage to such a synthetic tissue structure, and the ability to ensure sufficient passage of oxygen and nutrients to the cells of the synthetic structure. Also needed are synthetic structures and methods for generating synthetic structures where the patterning is consistent, reliable, the structure has sufficient strength and elasticity to survive in vivo for an extended period of time, and is easily retrievable. SUMMARY OF THE INVENTION

[0007] The present invention addresses the above-mentioned drawbacks of the prior art by using a manufacturing platform, means for suspending a bioprinted fiber structure, a bioprinting system incorporating the same, and a method of using the same to generate a tissue fiber structure having anti-FBR properties and improved structural stability. As first disclosed and demonstrated herein, 3D bioprinted fiber structures are generated in a manner that reduces or avoids contact with the receiving surface during printing, patterning, and / or processing. This can then significantly reduce or eliminate surface imprints or other defects, facilitate their transport and / or manipulation, and enable conformal coating with a material that can impart desired properties such as enhanced stability and / or anti-FBR across the outer surface of the bioprinted fiber structure.

[0008] Aspects of the present invention include a manufacturing platform for supporting a bioprinted fiber structure during printing, patterning, and / or processing, the platform including a frame that defines a void and includes a plurality of struts on both sides of the frame for fixing and suspending at least one crosslinkable fiber within the frame to form the fiber structure, wherein a continuous length of the at least one fiber is printed around at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the struts during a 3D bioprinting process.

[0009] In an embodiment, the strut is positioned inside the frame, and preferably, the strut is positioned on a frame protrusion extending into the cavity. In an embodiment, by relying on the protrusion, the contact between the fiber and the frame is minimized.

[0010] In an embodiment, the struts are arranged equidistantly or at non-uniform intervals around the frame.

[0011] In an embodiment, the frame preferably includes at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 struts having a height of about 0.5 mm to about 50 mm.

[0012] In an embodiment, at least a portion of the crosslinkable fiber contains a biologically-derived substance.

[0013] In an embodiment, the frame is triangular, rectangular, hexagonal, octagonal, circular, etc.

[0014] In an embodiment, the fiber structure includes a lattice.

[0015] In an embodiment, the frame is coupled to a mounting bracket configured to adjust the position of the frame relative to the receiving surface. In an embodiment, the frame further includes a fiber cutting groove positioned along the cavity to enable a cutting tool to cut a portion of the fiber structure. In an embodiment, the frame further includes a fitting groove disposed on the bottom surface of the frame and configured to receive the wall of a corresponding container or vessel on the receiving surface.

[0016] Aspects of the present invention include means for suspending a bioprinted fiber structure during printing, patterning, and / or processing, the means for suspension including an attachment bracket and / or a frame coupled to a receiving surface of a bioprinting system, the frame including a plurality of struts surrounding a frame for securing at least one crosslinkable fiber of a continuous length forming the fiber structure.

[0017] Aspects of the present invention include a bioprinting system. In embodiments, the bioprinting system includes a manufacturing platform disclosed herein or means for suspending a bioprinted fiber structure disclosed herein. In embodiments, the bioprinting system includes at least one dispensing orifice for dispensing the at least one crosslinkable fiber onto the receiving surface. In embodiments, the bioprinting system includes a positioning unit for positioning the receiving surface in three-dimensional space relative to the dispensing orifice, the positioning unit being operably coupled to either the receiving surface or the at least one dispensing orifice. In embodiments, the bioprinting system includes dispensing means for dispensing at least one crosslinkable fiber from the at least one dispensing orifice.

[0018] In embodiments of the bioprinting system, the manufacturing platform or the means for suspension is suspended on the receiving surface.

[0019] In embodiments, the receiving surface includes a porous material.

[0020] In an embodiment, the receiving surface includes a container containing a liquid, such as, for example, a crosslinking agent solution tank preferably positioned on or around a vacuum chuck or an optional dip coat. In an embodiment, the receiving surface includes a vacuum chuck and an integral container formed by a wall protruding from the upper surface of the vacuum chuck and defining the outer periphery of the integral container. In an embodiment, the wall of the integral container is preferably configured to be inserted into a mating groove on the bottom of the frame so as to fit snugly to prevent leakage of fluid beyond the wall when the frame is placed on the container.

[0021] In an embodiment, the bioprinting system further includes a programmable control processor for controlling the positioning components and for controlling the flow rate of one or more fluids via dispensing means.

[0022] In an embodiment, the dispensing means includes at least one pump, and optionally the at least one pump includes a pump assembly including a plurality of pumps radially positioned on a mounting bracket.

[0023] In an embodiment, the bioprinting system further includes at least one print head including a plurality of microfluidic printing channels for selectively providing a plurality of respective materials.

[0024] Aspects of the present invention include a method for bioprinting a fibrous structure. In an embodiment, the method includes providing a bioprinting system as disclosed herein and dispensing a continuous length of crosslinkable fiber around a plurality of the struts on the frame of the manufacturing platform to generate a fibrous structure.

[0025] In an embodiment, the method further includes applying a conformal coating to the entire outer surface of the fibrous structure while the fibers remain attached to the frame.

[0026] In an embodiment, the method further includes transporting the fibrous structure from one location to another while the fibrous structure remains attached to the frame.

[0027] In an embodiment, the method further includes storing the fibrous structure while the fibrous structure remains attached to the frame.

[0028] Aspects of the present invention include a bioprinted fibrous structure produced by the methods of the present disclosure. In an embodiment, the bioprinted fibrous structure includes a continuous length of crosslinkable fibers including at least one bioderived substance, the crosslinkable fibers including a solid core and at least one outer shell layer surrounding the solid core, and the bioprinted fibrous structure includes at least two layers of a lattice / grid formed by the continuous crosslinkable fibers.

[0029] In an embodiment, each layer has a thickness of from about 0.050 mm to about 3 mm.

[0030] In an embodiment, the bioprinted fibrous structure has a packing density between about 10% and about 90%, or between about 20% and about 80%, or between about 30% and about 70%, or between about 40% and about 60%, preferably about 30%, about 40%, about 50%, or about 60%.

[0031] In an embodiment, the solid core includes at least one bioderived substance and, optionally, the solid core is compartmentalized along the length of the fiber.

[0032] In an embodiment, the bioprinted fibrous structure includes at least one inner shell layer surrounding the solid core, the at least one inner shell layer including at least one bioderived substance, and, optionally, the solid core and / or the at least one inner shell layer are compartmentalized along the length of the fiber.

[0033] In an embodiment, the bioprinted fibrous structure includes at least one conformal coating.

[0034] In an embodiment, the solid core contains about 1.5% alginate, the at least one outer shell layer contains about 2.0% alginate, and the coating contains about 0.5% alginate.

[0035] In an embodiment, the bioprint fiber structure includes an inner conformal coating and an outer conformal coating.

[0036] In an embodiment, the biologically derived material includes pancreatic islet cells.

[0037] Other features, objects, and advantages will be apparent from the following disclosure.

[0038] Incorporation by Reference All publications, patents, and patent applications cited herein are hereby incorporated by reference in the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Brief Description of the Drawings

[0039]

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[0040] The means for suspension of the present disclosure advantageously enables continuous bioprinting of crosslinkable fibers such that the resulting structure can be suspended during one or more of printing, patterning, and / or post-printing processes. As demonstrated herein, the means and / or manufacturing platform including the same facilitate post-print modification, as well as storage and / or transport of bioprinted fiber structures. In embodiments, the present invention facilitates post-print modification such as coatings that can, for example, enhance stability and / or impart anti-FBR properties. In embodiments, the present invention enables the production of bioprinted fiber structures having a more uniform conformal coating that is free or substantially free of defects that may contribute to FBR when implanted within a subject. In embodiments, the fiber structures generated in accordance with the teachings of the present disclosure advantageously exhibit reduced FBR when implanted within a subject.

[0041] Definitions For the purpose of interpreting this specification, the following definitions apply, and where appropriate, terms used in the singular also include the plural and vice versa. If the definitions described conflict with the documents incorporated herein by reference, the definitions described below shall prevail. Unless otherwise stated, all technical and scientific terms used in this specification shall have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0042] As used herein, the term "hydrogel" refers to a composition comprising water and a network or lattice of hydrophilic polymer chains.

[0043] As used herein, the term "sheath fluid" or "sheath liquid" refers to a fluid that is at least partially used to enclose or "sheath" a material as it passes through a fluid channel. In some embodiments, the sheath fluid comprises an aqueous solvent such as water or glycerol. In some embodiments, the sheath fluid comprises a chemical crosslinking agent. Non-limiting examples of crosslinking agents include divalent cations (e.g., Ca 2+ , Ba 2+ , Sr 2+ etc.), thrombin, and pH-adjusting chemicals such as sodium bicarbonate.

[0044] As used herein, the term "segmentation / compartmentalization" refers to the discontinuous nature of the types of materials and / or biogenic substances contained in the core layer(s) and / or shell layer(s) of the fibers disclosed herein, e.g., there are intentional gaps in the deposition of biogenic substances along the length of the fiber. The spacing (e.g., length) between such segments / compartments may be regular (e.g., approximately the same spacing between regions of biogenic substances) or the spacing may vary.

[0045] As used herein, the term "solid core" refers to the core of a fiber of the present disclosure that consists of a particular material (e.g., a hydrogel that can be cross-linked by a chemical cross-linking agent), such that the core does not contain a lumen along the entire length of the fiber. The solid core of the present disclosure is not intended to refer to a core that is completely impermeable along its length, as it may allow the passage of certain fluids, molecules, and / or ionic species through the entire core.

[0046] As used herein, the term "annular fiber" refers to a fiber that consists of a solid core and one or more shell layers surrounding the solid core.

[0047] As used herein, the term "biocompatible material" refers to a material that can incorporate and / or come into contact with biological substances, including but not limited to cell-containing substances, and that does not exhibit a harmful effect on the ability of the biological substance to perform one or more functions (e.g., cell functions including, but not limited to, secretion of bio-related molecular species, agonist / receptor binding, signal transduction, etc.).

[0048] As used herein, the term "immunoprotective" broadly refers to a design aspect of a fiber of the present disclosure that helps to reduce, prevent, or eliminate a host immune response, such as immune cell invasion of the fiber, upon implantation of the fiber into the body (e.g., into a mammalian body).

[0049] As used herein, the term "agent" refers to any protein, nucleic acid molecule (including chemically modified nucleic acid molecules), antibody, small molecule, organic compound, inorganic compound, or other molecule of interest. Agents may include bio-related substances, therapeutic agents, diagnostic agents, pharmaceuticals, chelating agents, etc. A therapeutic agent or pharmaceutical is an agent that, when administered to a subject in a manner consistent with the present disclosure, either alone or in combination with additional compounds, elicits a desired response (e.g., a therapeutic or prophylactic effect). A bio-related substance is a substance that supports another biological process, e.g., supports the viability of cells.

[0050] Introduction 3D bioprinting is a layered manufacturing process in which synthetic fibers optionally containing cells are placed layer by layer to obtain a multi-layer 3D structure. Extrusion (Panwar A et al., Moleculars. (2016); 21:685; Sakai S et al., Biofabrication. (2018); 10:045007; Han HW and Hsu SH, Neral Regener.Res. (2017); 12:1595), inkjet (Gao G et al., Biotechnol. Lett. (2015); 37:2349; Gao G and Cui X, Biotechnol. Lett. (2016); 38:203, Bsoul A et al., Lab Chip. (2016); 16:3351), laser-assisted (Sorkio A et al., Biomaterials. (2018); 171:57; Pages E et al., J. Nanotechnol. Eng. Med. (2015); 6:021006; Cattros S et al., In Vivo and In Situ Biofabrication by Laser-Assisted Bioprintin, Elsevier, Winston-Salem, USA. (2015)), and stereolithography (SLA) (Miri AK et al., Adv. Mater. (2018); 30:1800242; Wang Z et al., ACS Appl. Mater. Interfaces. (2018); 10; 26859; Wang Z et al., Biofabrication. (2015); 7:045009) printing methods, various types of 3D bioprinting technologies have been developed. Among these, extrusion is the most common, by which bioink is dispensed through one or more syringes to form a layer-by-layer scaffold from the fibers.

[0051] With advancements, microfluidics-based 3D bioprinting systems have also come into use (Beyer ST et al., Transducers Eurosensors XXVII 17th Int.Conf.Solid-State Sensors,Actuators,Microsystems;IEEE, Piscataway, NJ (2013), pp.1206 - 1209; Beyer ST et al., The 17th Int.Conf.on Miniaturized Systems for Chemistry and Life Sciences (2013); pp.176 - 178). Using these systems and techniques, multiple materials (e.g., bioinks, crosslinkers, etc.) flow through microchannels, which can enable one or more precise controls such as flow rate, switching, mixing, etc. When used with a sheath flow surrounding at least one inner material, microfluidic bioprinting can reduce shear stress during the printing process. Microfluidics-based 3D bioprinting also advantageously allows for the intersection of material flows when the material flows exit independent flow paths and enter a single flow path (e.g., a dispensing channel), facilitating the generation of structures with a core surrounded by one or more shells.

[0052] Many of these bioprinting strategies print fibers directly onto the receiving surface, which can be a confounding factor from the perspective of post-print processing steps. For example, a coating cannot be applied to the bottom surface of a bioprinted fiber structure that remains in contact with the receiving surface, and manipulating the structure may cause the structure to collapse or otherwise reduce its integrity. Additionally, printing a fiber structure onto a surface can introduce unwanted defects or irregularities due to contact with the surface itself.

[0053] To explain the points, FIG. 1 shows an exemplary microfluidic print head 100 that can be used to print a fibrous structure 120. The print head 100 includes a plurality of reservoirs (104, 106, 108, 110) and corresponding valves (shown simply as "102"). The valves control the flow of material into their respective microfluidic channels, and the microfluidic channels converge into a single dispensing channel 122. In this exemplary figure, the microfluidic channel 112 directs a sheath fluid containing a cross-linking agent solution towards the dispensing channel 122, the channel 114 directs a buffer solution towards the dispensing channel 122, and the channel 116, which receives flow from one or both of the reservoirs 108 and 110, directs a hydrogel material towards the dispensing channel 122. Thus, reservoir 106 holds the sheath fluid, reservoir 104 holds the buffer solution, reservoir 108 holds a first hydrogel solution, and reservoir 110 holds a second hydrogel solution. Optionally, one or both of the first hydrogel solution and the second hydrogel solution contain cells. The flow of each material is controlled by valve 102.

[0054] Also shown in the example is a receiving surface 124 that includes a plurality of pores 125. In an exemplary method, the sheath fluid surrounds the hydrogel solution within the dispensing channel 122, such that cross-linking of the fibrous structure occurs while it is within the dispensing channel. Any excess sheath fluid flows through the receiving surface 124 as indicated by arrow 126, while the fibrous structure 120 is deposited on top of the receiving surface.

[0055] FIG. 1 shows a print head 100 for depositing a first layer of a fibrous structure 120. For example, additional layers can be added on top of the first layer to form a structure on a lattice / grid. As can be seen, the bottom surface of the fibrous structure is in contact with the receiving surface, and thus any procedure for coating the fibrous structure with a desired material will not be able to reach the bottom surface of the fibrous structure. In some cases, attempting to move and / or manipulate the fibrous structure to coat the bottom surface may result in a loss of fidelity of the fibrous structure due to a lack of effective adhesion between the fibers. Since the entire outer surface of the fibrous structure cannot be uniformly coated in this way (i.e., an additional conformal coat cannot be added), the fibrous structure may be unsuitable for implantation.

[0056] In some cases, due to the porous nature of the receiving surface, an imprint may occur in the structure along the length of the resulting fibers. As additional layers are added, the imprint / irregularity may become more pronounced due to an increase in the total weight of the bioprint structure. Even in the case of a non-porous surface, the weight of the additional layer may compromise the structural integrity of the bioprint fibrous structure. Further, in some cases, a vacuum may be relied upon to remove excess sheath fluid flowing through the pores 125 of the receiving surface 124, and the vacuum may exert a negative force on the fibers in the vicinity of the pores 125, which may further exacerbate the formation of the imprint / irregularity. Such an imprint / irregularity may contribute to FBR during implantation.

[0057] Means for suspending the bioprint structure This specification describes means for suspending a fibrous structure during printing and / or transport. In this way, the fibrous structure can be bioprinted such that the resulting structure is at least partially suspended during one or more of the printing process, the patterning process, and / or the post-printing process. In some examples, printing onto a receiving surface can be completely avoided. Referring to FIG. 2A, exemplary means for suspending a fibrous structure during printing are shown. In this exemplary embodiment, the means for suspension includes a frame 202 having a plurality of opposing struts 206. In embodiments, the frame and struts 206 are made of a single fabrication material, although it is within the scope of the present disclosure that the struts 206 may be made of a different material than the frame 202. In embodiments, the frame 202 and / or the struts 206 are made of, for example, stainless steel or a dental grade polymer (e.g., polyethylene, polymethylmethacrylate, polycarbonate, polyethylene glycol, polyurethane, hexamethyldisilazane, etc.). In embodiments, the choice of the material composition of the struts relates to minimizing or optimizing the level of adhesion of the fibrous structure to the struts.

[0058] As shown in FIG. 2A, the empty frame 202 defines a void and includes, on both sides of the frame, a plurality of opposing struts 206 that extend upwardly substantially perpendicular to the frame 202. In the illustrated exemplary embodiment, the struts 206 are positioned inside the frame 202, more specifically on protrusions of the frame 202 that extend beyond the void, so as to minimize contact between the fibers and the frame 202 during the printing process. In the embodiment shown in FIG. 2A, the frame 202 has a generally square shape, but other frame shapes including, but not limited to, rectangular, circular, triangular, hexagonal, and octagonal are readily contemplated as being within the scope of the present disclosure. In embodiments, the struts may be equally spaced along all sides of the frame, although the spacing may also be irregular and / or the opposing struts may be positioned on only two sides of the frame depending on the application. At least, the plurality of struts 206 are positioned on both sides of the frame and are spaced sufficiently apart to allow a needle or dispensing nozzle to move around / through the struts when the fiber structure is being printed.

[0059] The number of struts included within the frame for suspending the fiber structure during printing can vary and may be a function of several variables including, but not limited to, the dimensions of the corresponding frame (e.g., outer perimeter length), the size of the dispensing needle / nozzle, the desired application, the strut thickness, the strut shape (e.g., cylindrical, square, semi-cylindrical), etc. For example, the frame may include at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or more than 50 struts, e.g., 100 or more struts. The strut thickness (e.g., diameter in the case of a cylindrical or semi-cylindrical strut) and the strut height are additional variables that can be selected depending on the desired application. For example, the strut height may vary between about 0.1 mm and about 10 cm, preferably between about 0.2 mm and about 20 mm, e.g., between about 1 mm and about 4 mm.

[0060] Referring to FIG. 2B, another exemplary embodiment of the frame 210 is shown. The frame 210 includes struts 212 along with corner struts 214. The frame 210 has a greater number of struts (11 struts on each side) along sides 220 and 221, and a lesser number of struts (9 struts on each side) along sides 222 and 223. In these aspects, the frame 210 is substantially similar to the frame 202 (see FIG. 2A). In other examples, each side may have the same number of struts. Further, although shown as having two struts at two opposing corners, other embodiments include struts at all four corners, only three corners, only one corner, or no corners.

[0061] In an embodiment, the struts are positioned on frame protrusions that extend inwardly from the frame into the interior of the frame. Referring particularly to FIG. 2B, each strut (e.g., 212 and 214) is positioned on a protrusion 216 that extends inwardly from the frame 210. In FIG. 2B, each protrusion 216 is triangular, but the protrusions 216 that support the struts (e.g., 212 and 214) may be of other shapes (e.g., rectangular, square, semi-circular). In some embodiments, the struts (e.g., 212, 214) can be attached directly to the interior of the frame (e.g., frame 210) instead of the protrusions 216. The frame 210 includes a handle 218 that can be used to grip (e.g., manually or robotically) the frame 210 and manipulate / move the frame 210.

[0062] As shown in FIG. 2B, the frame 210 has a generally bowl shape with a handle 218 affixed to the upper edge 230 and protrusions 216 extending from the lower edge 232. In an embodiment, the overall bowl shape can enable the frame 210 to be conveniently installed in a corresponding attachment structure, as will be described in more detail below.

[0063] For reference, another frame 250 is shown in FIG. 2C. Clearly shown are the upper edge 252 and the lower edge 254. The struts 256 each extend from a protrusion 258 that extends inward from the lower edge 254. The handle 260 extends from the upper edge 252. In the example shown in FIG. 2C, each side has the same number of struts, and the corner struts 262 occupy two corners of the frame 250.

[0064] Depending on the frame, the bioprinted fiber structure of the present disclosure may have different packing densities. The packing density described herein is expressed as a percentage. For example, a lattice structure having a completely filled fiber structure (i.e., no space) would correspond to a packing density of 100%, while a lattice structure that is not occupied by any fiber structure by 90% would correspond to a packing density of 10%. What is related to the packing density is the distance between the fibers. The inter-fiber distance described herein refers to the distance of the empty space between two adjacent fibers in a layer of a lattice where the fibers in the same layer are parallel to each other (see, for example, the exemplary fiber structure shown in FIG. 4).

[0065] The frames shown in FIGS. 2A-2C are exemplary, and other frame designs are within the scope of the present disclosure. FIG. 2D shows another frame 275 in which the struts 276 are arranged at irregular intervals. The frame 275 includes a common outer envelope 277 and a void space 278. The frame 275 is shown from a top view.

[0066] In some embodiments, the frame can include a fiber cutting groove positioned so that a cutting instrument, such as a surgical scalpel, can cut a portion of the fiber structure, such as long initial fibers that may be "waste." The fiber cutting groove can enable alignment of the cut without the need to remove the fiber structure before cutting the "waste" portion of the fiber. An exemplary frame 280 having a fiber cutting groove 282 is shown in FIG. 2E.

[0067] Accordingly, as shown with respect to FIGS. 2A - 2E, the frame according to the present disclosure may be of a particular shape (e.g., square, rectangular, triangular, hexagonal, octagonal, circular, irregular, etc.). In some examples, the struts (e.g., strut 206 in FIG. 2A) may be spaced equidistantly around the frame, as illustratively shown in FIGS. 2A - 2C. However, in other examples, the struts (e.g., strut 276 in FIG. 2D) may be spaced irregularly around the frame. In an embodiment, the struts are positioned on frame protrusions extending internally from the frame, preferably within a cavity formed by the frame, inside the frame so as to minimize contact between the fibers and the frame during the printing process. In an embodiment, the overall shape of the frame may be unique for use in conjunction with a particular mounting bracket, as detailed more specifically below. Accordingly, in some embodiments, the frame can print a fiber structure consisting of a regular lattice structure. In some embodiments, the frame can be used to print a fiber structure consisting of an irregular lattice structure.

[0068] Referring to FIG. 3, a print head 302, a support column 306, a frame 308, a protrusion 310, a dispensing channel 314, and a bioprint fiber structure 316 are shown. As shown, since the fiber structure can be printed around the support column 306, it becomes possible to suspend the fiber structure 316 within the frame during printing, patterning, and / or post-printing processes. In FIG. 3, only two opposing support columns (left and right) are shown for illustrative purposes. In the exemplary example shown in FIG. 3, the fiber structure 316 is a grid / lattice structure consisting of a first layer 320, a second layer 322, and a third layer 324 (being formed). Layer 322 is shown in cross-section and the layers are formed by continuously printing around opposing support columns of the frame 308. Support columns (e.g., 306 in FIG. 3) may further serve to impart tension to the fibers as they are printed. This may help to ensure substantial linearity of the fibers between the support columns and, in some examples, may help to maintain a desired distance between adjacent fiber segments. In some embodiments where a grid / lattice is printed using a frame such as the frames shown in FIGS. 2A - 2C, the distance of the empty space between adjacent fibers, referred to herein as the fiber spacing, may be between about 1000 μm and 2000 μm, for example, between about 1400 μm and about 1600 μm.

[0069] Referring to FIG. 4, a frame 202 and struts 206 are shown along with an exemplary fiber structure 410. The fiber structure 410 is shown wrapped around each of the opposing struts 206 in a way that generates a bio-printed lattice structure suspended within the frame. To generate the fiber structure, a first layer can be generated by moving fibers back and forth in the direction of arrow 414 around successive opposing struts 206, and then a second layer can be generated by again moving fibers back and forth in the direction of arrow 418 around successive opposing struts 206, or vice versa. In principle, any number of layers can be added in such a way. In embodiments, the height of the struts 206 may vary as a function of the number of layers desired for a particular structure, and the greater the height of the struts, the greater the potential number of layers. Since the height of the layers described herein is a function of the diameter of the fibers, a structure composed of smaller diameter fibers will have more layers than a structure composed of larger diameter fibers when the overall height of the bio-printed fiber structure is the same. As one representative example, a structure 10 mm in height composed of fibers 0.050 mm in diameter will have 200 layers.

[0070] Preferably, the fiber structure is generated by wrapping a continuous length of fiber around at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, e.g., 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more struts during printing of the fiber structure.

[0071] As shown in FIG. 4, when generating the fiber structure 410 in the manner described, loops 420 are obtained. In embodiments, the loops may be removed from the post-print of the fiber structure or may remain as part of the fiber structure. In embodiments where the loops are cut, the loops can be cut while the fiber structure otherwise remains attached to the frame, or the fiber structure can be removed from the frame and then the loops can be cut.

[0072] In an embodiment, to generate a bioprinted fiber structure, by relying on a frame as shown in FIGS. 2A-4, it is possible to manipulate the entire fiber structure without disturbing the structural integrity of the fiber structure before and / or during the adhesion between fibers within a layer. Thus, the bioprinted fiber structure coupled to the frame can be lifted above a surface (e.g., a receiving surface) and can cover the entirety thereof. In an embodiment, removing a fiber structure (e.g., a fiber structure substantially similar to fiber structure 410) can be accomplished simply by inverting the frame such that gravity acts on the fiber structure to release it from the frame. In some additional or alternative embodiments, while the frame is inverted or at least partially inverted, a force can be applied to the underside of the frame to assist in releasing the fiber structure from the frame.

[0073] Manufacturing platform The manufacturing platform for generating the bioprinted fiber structure disclosed herein includes means for suspending at least a bioprinted fiber structure as described above (e.g., frame 202 of FIG. 2A, frame 210 of FIG. 2B, frame 250 of FIG. 2C, frame 275 of FIG. 2D, frame 280 of FIG. 2E), including a plurality of struts for fixing at least one bioprinted fiber to form the bioprinted fiber structure. For example, in an embodiment, the means for suspending the bioprinted fiber structure is a stand-alone device that can be used, for example, in conjunction with a particular bioprinter system. In other embodiments, one or more additional components can be included as part of the manufacturing platform.

[0074] Referring to FIG. 5A, a manufacturing platform 500 is shown. The manufacturing platform 500 includes a lifting arm 502 coupled to a mounting bracket 506. The mounting bracket 506 is configured to receive a frame 508 (e.g., similar or the same as the frames shown in FIGS. 2A - 2D). The lifting arm 502, and alternatively the mounting bracket 506 and the frame 508, can be adjusted manually or robotically (i.e., in an automated manner) to adjust the height at which the frame 508 is positioned relative to the surface 510. In embodiments, the lifting arm 502 is adjustable between a finite number of positions (e.g., 2, 3, 4, 5, 6, 8, 10). In embodiments, the lifting arm 502 is adjustable between any number of positions. In either case, the lifting arm 502 can be fixed to prevent further movement of the lifting arm until further adjustment is desired with the lifting arm 502 set to the desired position.

[0075] In embodiments, the manufacturing platform includes a vacuum chuck 512. The vacuum chuck 512 includes a chuck orifice 516 that can be used in conjunction with a vacuum source (e.g., a vacuum pump) to draw a vacuum inside the vacuum chuck 512. The vacuum chuck 512 can be placed on the surface 510 and aligned with the frame 508 and the mounting bracket 506. In embodiments, the upper portion of the vacuum chuck 512 is of an area that is substantially the same as or larger than the area corresponding to the inside of the frame 508. In embodiments, the upper portion of the vacuum chuck 512 is porous, such that the vacuum applied to the vacuum chuck 512 through the chuck orifice 516 draws air into the internal space of the vacuum chuck 512 through the upper portion of the vacuum chuck 512. In embodiments, the upper portion of the vacuum chuck 512 is uniformly porous throughout the upper portion.

[0076] In an embodiment, the mesh 514 can be placed on top of the vacuum chuck 512. The mesh 514 can be reusable or disposable. Preferably, the mesh 514 is made of a non-adhesive, non-reactive synthetic material that can function as a passive support for the bioprinted fiber structure in some examples. Examples include, but are not limited to, nylon, polyethylene, polyethylene terephthalate, steel, glass, PEEK (polyetheretherketone), PTFE (polytetrafluoroethylene), cellulose, and the like.

[0077] In an embodiment, for example, the frame 508 can be lifted away from the surface 510 via the lifting arm 502 to enable placement of the frame 508 at a desired position relative to the frame 508 of the vacuum chuck 512 (and optionally the mesh 514). Next, as illustratively shown in FIG. 5B, the frame 508 can be lowered in the direction of the surface 510 via the lifting arm 502 to position the frame 508 at a desired position relative to the vacuum chuck 512 (and optionally the mesh 514). It can be understood that the vacuum drawn through the vacuum chuck 512 can help remove excess fluid (e.g., excess sheath fluid and / or excess buffer fluid) that might otherwise accumulate on the mesh 514, on top of the vacuum chuck 512, and / or on the surface of the bioprinted fiber structure. Thus, in the embodiments shown in FIGS. 5A-5B, the vacuum chuck 512 includes a fluid removal component.

[0078] The mounting bracket (e.g., mounting bracket 506) and optionally the lifting arm (e.g., lifting arm 502) can be designed to accommodate frames of a specific size. In some embodiments, the mounting bracket and the lifting arm are a single unit, while in other embodiments, the mounting bracket can be detachably coupled to the lifting arm.

[0079] Accordingly, for reference, FIG. 5C shows another exemplary embodiment of a manufacturing platform 522 having a lifting arm 524 in a first upper position, and FIG. 5D shows a manufacturing platform 522 having a lifting arm 524 in a second lower position. Mounting brackets 526 as shown in FIGS. 5C-5D are designed to accommodate a frame 528. The manufacturing platform 522 can also include a vacuum chuck and a mesh (not numbered), as described above with respect to FIGS. 5A-5B. Visibly, the manufacturing platform 522 accommodates a smaller frame than the manufacturing platform 500. In some embodiments, the mounting bracket(s) can be detachably coupled to the lifting arm of a particular manufacturing platform to accommodate different frame calibrations and / or dimensions.

[0080] In an embodiment, one or more homing struts 520 are included as part of the mounting bracket 526, as shown in FIG. 5D. The homing posts 520 can be used, as disclosed herein, for example, to align the print head of a bioprinting system. The homing struts 520 function, for example, as a reference coordinate that can assist a bioprinter system in finding and setting a "home" position for printing a 3D fibrous structure. For example, the home position can correspond to known x, y, z coordinates relative to the homing struts 520. In this way, the bioprinting system can register the dimensions of a particular frame and adjust the movement of the print head as a function of the reference coordinates.

[0081] In an embodiment, at least one continuous length of a bioprint fiber structure is printed around at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, for example 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more of the struts of a frame (e.g., frame 508) during a 3D bioprinting process by using a manufacturing platform as disclosed. Such a manufacturing platform can include part of a bioprinting system as disclosed herein.

[0082] In an embodiment, the upper portion of the mesh (e.g., mesh 514 in FIGS. 5A-5B) and / or the vacuum chuck (e.g., vacuum chuck 512) can include a receiving surface for the bioprinted fiber structure printed via a support corresponding to the frame (e.g., frame 508). In an embodiment, the receiving surface can include a surface (e.g., surface 510) that does not include, for example, the vacuum chuck (e.g., vacuum chuck 512), but optionally can include other additional or alternative fluid removal components.

[0083] Thus, in an embodiment, the receiving surface is flat or substantially flat. In an embodiment, the receiving surface is solid. In an embodiment, the receiving surface is porous. In an embodiment, the fiber structure is printed on the receiving surface (e.g., mesh 514) via the frame (e.g., frame 508), and then the frame and the corresponding fiber structure are suspended above the receiving surface for further processing, such as application to one or more coats of fiber structure.

[0084] In some embodiments, the manufacturing platform can include a container (see, e.g., FIG. 7). In an embodiment, the container can include a container in which means for suspending at least the bioprinted fiber structure (e.g., frame 508 in FIG. 5A), optionally coupled to an attachment bracket (e.g., attachment bracket 506 in FIG. 5A), can be installed within at least a portion of the manufacturing platform. For example, the container can include a solution tank used during one or more of printing, patterning, and / or modifying the bioprinted fibers after printing. In an embodiment, the container is sized such that the liquid solution held by the container can completely immerse means for suspending at least the bioprinted fiber structure (e.g., frame 508 in FIG. 5A), optionally coupled to an attachment bracket (e.g., attachment bracket 506 in FIG. 5A). In an embodiment, such a container can be used for submerge bioprinting as described below.

[0085] In an embodiment, the integrated container 530 can be formed directly on the upper surface of the vacuum chuck 532 by a wall 534 that defines the outer periphery of the container, as shown in FIG. 5E. The region surrounded by the wall can hold a liquid such as, for example, a crosslinking agent solution, a cleaning solution, a storage solution, an immersion coating solution, etc., and an integrated container can be created on which a frame 536 can be installed. In an embodiment, the wall is configured to interfere with the fitting groove 538 below the frame 536, as shown in FIG. 5F. Preferably, the depth of the groove is such that the volume of liquid in the container can be located just above the height of the struts of the frame. By having a liquid volume surrounded by the wall, the user can accurately dispense a specific amount of liquid onto the frame.

[0086] This configuration enables the fiber structure to be suspended in the liquid within the container without contacting the vacuum chuck and / or the mesh. In an exemplary embodiment, the volume of liquid surrounded by the walls may be sufficient to coat at least the lower side of the fiber structure when the frame is placed on the integrated container, and additional liquid may optionally be applied on top, as shown in FIG. 5G. In an embodiment, the vacuum chuck can be operably connected to a valve (540), and the valve (540) can be operated to allow the liquid to flow out of the container under vacuum or to prevent the flow of liquid to maintain the liquid level within the container.

[0087] In an embodiment, the frame or a part thereof can be coated with a hydrophobic coating such as Parylene-C to prevent leakage. In an embodiment, as shown in FIG. 5H, a vacuum chuck (542) having an integrated container can be lifted onto a support and used as a stand-alone immersion coating station for the frame (544).

[0088] The manufacturing platforms shown in FIGS. 5A - 5D are exemplary, and other variations are within the scope of the present disclosure. Referring to FIG. 5I, another example of a manufacturing platform 570 that may include a part of the 3D printing system disclosed herein is shown. The manufacturing platform 570 includes a frame 571, a vacuum chuck 572, and a fixed dock 574 that also includes a frame dock 575. An attachment bracket 577 is coupled to the frame 571. The vacuum chuck 572 includes pegs 576. A stage 573 is also shown. The 3D printing system can move the stage 573 along the x, y, and z planes (see Cartesian coordinate system 579). The vacuum chuck 572 may be placed on the stage 573 or may be fixed to the stage 573 in other cases. The frame dock 575 can be removably coupled to the frame 571, so the frame dock holds the frame 571 in place when the frame 571 is not coupled to the vacuum chuck 572 via the attachment bracket 577. In the figures of this example, the peg 576 coupled to the vacuum chuck 572 is disposed at the top of the groove 578, and as a result, the frame 571 is positioned directly above the vacuum chuck 572.

[0089] Referring to FIG. 5J, a manufacturing platform 570 is shown where the frame 571 is coupled to the frame dock 575 and the stage 573 is away from the frame 571. FIG. 5K shows the manufacturing platform 570 where the stage 573 has moved in such a way as to position the peg 576 within the bottom groove of the attachment bracket 577. By doing so, the frame 571 is effectively suspended above the vacuum chuck 572 (Δh). This allows the 3D printed fiber structure to be fully suspended for post - printing processes such as coating the entire 3D printed fiber structure. With respect to FIGS. 5I - 5K, the movement of the stage 573 can be automated, and as a result, the control system of the 3D printing system can effectively position the frame at a desired distance from the vacuum chuck (or other receiving surface) via a programmed control sequence.

[0090] Immersion bioprinting In embodiments, the fibrous structure can be printed into a solution bath containing a crosslinking agent solution, and the solution bath includes means for suspending the fibers during printing. FIG. 6 shows an exemplary depiction of such a process. As shown, FIG. 6 includes a print head 602, struts 606 (only two of which are shown for clarity) included as part of the means for suspending the bioprinted fibrous structure, a surface 610, and a container 612 located above the surface 610 and holding the crosslinking agent solution 620. In some embodiments, as described in more detail below, the surface 610 can include the upper surface of a vacuum chuck (e.g., the vacuum chuck 512 of FIGS. 5A-5B). In this way, the bioprinted fibrous structure 616 emerging from the dispensing channel 614 can be effectively crosslinked on all sides immediately after / during its dispensing. Further, the fibers can be completely or at least partially suspended above the bottom surface 630 during at least a portion of the printing process. Printing into a solution bath containing a crosslinking agent solution can reduce the effect of gravity on the bioprinted fibrous structure that is completely or at least partially suspended, which can help avoid weight-induced distortion and maintain the structural integrity of the bioprinted structure. In additional or alternative examples, printing a completely or at least partially suspended fibrous structure in a solution bath containing a crosslinking agent solution can prevent the introduction of imprints / irregularities into the bioprinted fibers that may contribute to the FBR when implanted into a subject.

[0091] In an embodiment, after the generation of a desired bioprinted fiber structure, the crosslinking solution can be removed. In some embodiments, removal of the crosslinking solution includes aspirating or otherwise draining the solution from within the container (e.g., via a removably plug positioned appropriately). In such embodiments, one or more additional fluids can then be added to the container for further processing and / or storage purposes. For example, one or more washing steps can be performed, where after removal of the crosslinking solution, a wash buffer is added to the container and then removed, and this process can be repeated any number of times. In some additional or alternative embodiments, the means for suspending the bioprinted fiber structure can be removed from the crosslinking solution along with the attached bioprinted fiber structure. In such an example, the process of removing the means for suspending the bioprinted structure can be manual or automated. In an embodiment, when the means for suspending the bioprinted fiber structure is removed from the crosslinking solution, the attached bioprinted fiber structure can optionally be further processed. In one embodiment, the means, and the attached bioprinted fiber structure can be placed within another container holding another solution for further processing and / or storage purposes. Other additional or alternative processing steps are described in further detail below. Importantly, the means for suspending the bioprinted fiber structure enables the fiber structure to be moved without disturbing its structural integrity.

[0092] Referring to FIG. 7, a manufacturing platform 700 is shown that includes a lifting arm 702, a mounting bracket 704, a frame 706, a vacuum chuck 708, a surface 710, and a container 720. In the embodiment shown in FIG. 7, the container 720 is positioned above the vacuum chuck 708. In this way, the mounting bracket 704 and the frame 706 can be lowered into a solution (not specifically shown) contained within the container 720. In some embodiments, the solution can include the cross-linking agent solution described, and optionally, the fibrous structure can be printed onto the frame 706 while the frame is immersed in the cross-linking agent solution. In additional or alternative embodiments, the bioprinted fibrous structure need not necessarily be printed in the solution, but the bioprinted fibrous structure attached to the frame can be lowered into the solution contained within the container after printing. The solution can be, for example, a cross-linking agent solution, a cleaning solution, a preservation solution, or in some embodiments, an immersion coating solution (described in more detail below).

[0093] In some embodiments, by placing a container (e.g., container 720 of FIG. 7) above a vacuum chuck (e.g., vacuum chuck 708 of FIG. 7), the frame (e.g., frame 706 of FIG. 7) can be easily lifted out of the solution contained within the container, the container can then be removed from above the vacuum chuck, and the frame can then be lowered, depending on the associated vacuum, for removal of excess fluid associated with one or more of the bioprinted fibrous structure, the frame, and / or the mounting bracket, in proximity to the top of the vacuum chuck. Such a process can be repeated any number of times as so desired.

[0094] Conformal coating As described herein, the coating process can add one or more additional outer layer(s) to the bioprinted fiber structure. Advantageously, the present invention allows the entire printed fiber structure, including its bottom surface, to be uniformly coated one or more times. In embodiments, these one or more additional outer layer(s) can impart anti-FBR properties and / or enhanced stability to the bioprinted fiber structure.

[0095] Referring now to FIG. 8, an exemplary process for adding a conformal coat to the bioprinted fiber structure of the present disclosure is shown. In the exemplary process flow of FIG. 8, the bioprinted fibers forming the fiber structure are generated such that the fibers are thereby exposed to a sheath fluid containing a crosslinking agent during printing. It should be understood that the bioprinted fiber structure described in the context of FIG. 8 can include a grid / lattice structure generated via means for suspending the bioprinted fiber structure (e.g., the frame shown in FIGS. 2A-2D), as disclosed herein.

[0096] Step (1) of FIG. 8 involves printing a fibrous structure. In this example, the fibers that make up the fibrous structure include a core 802 and an outer shell layer 804 surrounding the core, and the core is then surrounded by a sheath fluid 806 containing a crosslinking agent during printing. In embodiments, the outer shell layer 804 and the optional core 802 include crosslinkable materials. In embodiments, the core 802 is solid and optionally further includes at least one biogenic substance (e.g., cells). Although shown as one outer shell layer, bioprinted fibers including multiple shell layers are within the scope of the present disclosure. As the fibrous structure is printed onto a means for suspension, the sheath fluid is removed, for example, by flowing through the porous receiving surface (not shown) described above. After printing, optional step (2) includes immersing the entire printed fibrous structure 810 in a crosslinking solution 812 to facilitate / continuing uniform crosslinking of the entire printed fibrous structure. Although not explicitly shown, in other additional or alternative embodiments, for example, by means for suspension (e.g., the frames shown in FIGS. 2A-2D), the crosslinking solution can be dispensed onto the fibrous structure while it remains suspended, thereby washing the entire fibrous structure with the crosslinking solution.

[0097] Step (3) is divided into two sub-steps (3a) and (3b). Step (3a) includes coating the entire fibrous structure 810 with a coating solution 816. Such coating can include, for example, dispensing the coating solution 816 onto the fibrous structure while the fibrous structure is suspended by means for suspending the fibrous structure and / or immersing it in the coating solution 816. In embodiments, the coating solution 816 includes a crosslinkable material (e.g., alginate). In embodiments, the coating solution 816 includes the same material as the material of the outer shell layer 804 of the fibrous structure 810 being coated. In embodiments, the coating solution 816 includes a material different from the material of the outer shell layer 804 of the fibrous structure 810 being coated. In step (3a), residual crosslinking agent (e.g., Ca 2+) contributes to the initial cross-linking of the materials in the coating solution 816 and the materials including the outer shell layer 804. After the conformal coating of the fibrous structure 810, step (3b) includes immersing (or otherwise cleaning) the coated fibrous structure 818 in the cross-linking solution 812 / with the cross-linking solution 812. In this way, the conformal coat layer 820 is uniformly added to the entire fibrous structure as shown.

[0098] In embodiments, multiple coatings can be successively added to the fibrous structure. For example, adding 2, 3, 4 or more conformal coating layers successively to the bioprinted fibrous structure is within the scope of the present disclosure. By using the present invention, the fibrous structure can be suspended during the application of each conformal coating layer, so that the entire outer surface of the resulting structure can be coated any number of times. In embodiments, the thickness of the coating layer can be visually determined, for example, using microscopy.

[0099] In embodiments, Ca 2+ The chelating agent is applied immediately prior to step (3a) to remove some amount of Ca 2+ from the surface of the fibrous structure prior to the application of the conformal coating to improve the adhesion between the fibrous structure and the coating layer. Preferred examples of calcium chelating agents include, but are not limited to, BAPTA, EDTA, trisodium citrate, and their derivatives or analogs. A similar process may be used to successively add multiple conformal coatings to the bioprinted fibrous structure.

[0100] In an embodiment, the conformal coating can be selected from the perspective of material composition to impart specific properties. Different coating layers may, for example, consist of different materials and / or have different physical properties (e.g., different hardness levels). In an exemplary example, the first conformal coating and the second conformal coating may each consist of alginate, but the percentages are different. For example, the first conformal coating may consist of a higher percentage of alginic acid (e.g., about 2%), while the second coating may consist of a lower percentage of alginic acid (e.g., about 0.5%). Without being bound by theory, such an approach may impart enhanced stability due to the high percentage of alginate in the first coating, while at the same time imparting anti-FBR properties due to the low percentage of alginate in the second coating (Doloff et al. Nat. Biomed. Eng. (2021); 5(10):1115-1130).

[0101] Accordingly, the conformal coating enabled herein can advantageously be used to impart stability to the tissue fiber structure and / or to impart anti-FBR properties. According to the present invention, no part of the bioprinted fiber structure lacks the specific properties imparted by one or more coating layers.

[0102] In an embodiment, the coating layer (e.g., 820) is composed of a hydrogel material, such as a hydrogel material containing one or more of alginate, chitosan, GEL-MA, poly(ethylene glycol) (PEG), poly-L-lysine (PLL), triazole, etc. In some examples, the coating layer is composed of a functionalized alginate, i.e., an alginate chemically modified to contain one or more properties including, but not limited to, immunoprotective properties, which are advantageous in the production of the fibrous structures of the present disclosure. Examples of functionalized alginates include, but are not limited to, methacrylated alginate, alginate furan, alginate thiol, alginate maleimide, and covalent click arginate (e.g., alginate hydroxide (DMPS)-aldehyde (DMAPS-Ald) blended with [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium and / or [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (DMAPS)-hydrazide (DMAPS-Hzd)) (see U.S. Provisional Patent Application No. 63 / 192552).

[0103] In an embodiment, the coating solution contains at least one crosslinkable material, such as a hydrogel, e.g., alginate, zwitterionic alginate, sulfobetaine methacrylate (SBMA), chitosan, poly(ethylene glycol) diacrylate (PEGDA), poly(-ethylene glycol)-tetraacrylate (PEGTA), hyaluronic acid (HA), hyaluronic acid methacryloyl (HAMA), collagen, methacrylated collagen (ColMA), gelatin, gelatin methacryloyl (gelMA), agarose, gellan, fibrin (fibrinogen), poly(vinyl alcohol)) (PVA), etc., or any combination thereof, but not limited thereto.

[0104] Input material Aspects of the present invention include feedstock materials that can be used to print fibrous structures for advantageous use as biomaterials. As used herein, "biomaterial" refers to natural or synthetic substances that are useful for constructing or replacing tissues, such as human tissues, with or without living cells. In the field of bioprinting, the term "biomaterial" is often synonymous with the term "biolink".

[0105] The feedstock materials generally include at least one crosslinkable material, such as alginate, chitosan, PEGDA, PEGTA, hyaluronic acid (HA), HAMA, collagen, CollMA, gelatin, GelMA, agarose, gellan, fibrin (fibrinogen), PVA, etc., or any combination thereof, including but not limited to hydrogels, as well as PCL, poly-(d,l-lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), etc., or any combination thereof, including but not limited to preferred hydrogels. In a preferred embodiment, the feedstock material includes at least one hydrogel. Non-limiting examples of hydrogels include alginate, agarose, collagen, fibrinogen, gelatin, chitosan, hyaluronic acid-based gels, or any combination thereof. Various synthetic hydrogels are known and can be used in embodiments of the systems and methods provided herein. For example, in some embodiments, one or more hydrogels form at least a portion of the structural basis of the three-dimensional structure to be printed. In some embodiments, the hydrogel has the ability to support the growth and / or proliferation of one or more cell types, and one or more cell types can be dispersed within the hydrogel or added to the hydrogel after being printed in a three-dimensional configuration.

[0106] In some embodiments, the hydrogel is crosslinkable by a chemical crosslinking agent. For example, a hydrogel containing alginate can be crosslinkable in the presence of a divalent cation such as calcium chloride, a hydrogel containing chitosan can be crosslinked using a polyvalent anion such as sodium tripolyphosphate (STP), a hydrogel containing fibrinogen can be crosslinkable in the presence of an enzyme such as thrombin, and a hydrogel containing collagen, gelatin, agarose, or chitosan can be crosslinkable in the presence of heat or a basic solution.

[0107] In embodiments, the hydrogel fibers can be produced by a precipitation reaction achieved by extracting the solvent from the input material upon exposure of the input material to a crosslinking agent material that is miscible with the input material. Non-limiting examples of input materials that form fibers via a precipitation reaction include collagen and polylactic acid (PLA). Non-limiting examples of crosslinking materials that enable precipitation-mediated hydrogel fiber formation include polyethylene glycol (PEG) and alginate. Crosslinking of the hydrogel increases the hardness of the hydrogel and, in some embodiments, enables the formation of a solidified hydrogel.

[0108] In some embodiments, the hydrogel contains alginate. Alginate forms a solidified colloidal gel (high water content gel or hydrogel) upon contact with a divalent cation. Any suitable divalent cation can be used to form a solidified 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+ , Ca 2+ has the optimal characteristics and is most commonly used (Ouwerx, C. et al., Polymer Gels and Networks, 1998, 6(5): 393 - 408). Studies have shown that Ca 2+It has been shown that through the cooperative binding of ions, a calcium alginate gel, the so-called "egg box" model, is formed (ISP Alginates, Section 3: Algin-Manufacture and Structure, in Alginates: Products for Scientific Water Control, 2000, International Specialty Products: San Diego, pp. 4-7). G-rich alginates are thermally stable and tend to form strong but brittle Ca gels, while M-rich alginates are less thermally stable and tend to form weaker but more elastic gels. In some embodiments, the hydrogel comprises depolymerized alginate.

[0109] In some embodiments, the hydrogel is crosslinkable using a free radical polymerization reaction, and covalent bonds are formed between molecules. Free radicals can be generated by exposing a photoinitiator to light (usually ultraviolet light), or by exposing the hydrogel precursor to a chemical source of free radicals such as ammonium persulfate (APS) or potassium peroxodisulfate (KPS) combined with N,N,N,N-tetramethylethylenediamine (TEMED) as initiator and catalyst, respectively. Non-limiting examples of photocrosslinkable hydrogels include methacrylated hydrogels such as methacryloyl hyaluronic acid (HAMA), gelatin methacrylate (GEL-MA), or polyethylene (glycol) acrylate-based (PEG-acrylate) hydrogels, which are used in cell biology because they are inert to cells. Polyethylene glycol diacrylate (PEG-DA) is commonly used as a scaffold in tissue engineering because polymerization occurs rapidly at room temperature, requires little energy input, has a high water content, is elastic, and can be customized to contain various biomolecules.

[0110] In embodiments, the input material includes a non-biodegradable polymer. In an example, the input material may be a synthetic polymer, such as polyvinyl acetate (PVA). In embodiments, the input material may include hyaluronic acid (HA).

[0111] In some embodiments, the hydrogel includes a chemically modified alginate. In an example, the chemically modified alginate includes an alginate functionalized with methacrylate groups, which is referred to herein as “Alg-MA”. In some embodiments, Alg-MA can be used in an immune defense shell layer by blending with a zwitterionic alginate, referred to herein as “Alg-zw”. Since Alg-MA has a dual crosslinking ability, in embodiments, Alg-MA can first be printed with Alg-zw via physical crosslinking. Next, during printing, the fibers can be further irradiated to induce covalent crosslinking between the fibers, thus generating F-F adhesion. In some embodiments, the chemically modified alginate may include a thiolated alginate.

[0112] In some embodiments, one or more synthetic components can be added to the hydrogel material. The synthetic components may be useful in improving inter-fiber adhesion and / or in vivo stability. In an example, the material may include an acrylated zwitterionic monomer (e.g., sulfobetaine methacrylate (SBMA)) and a crosslinking agent (e.g., poly(ethylene glycol) diacrylate (PEGDA)). In such an example, photo-mediated crosslinking of the zwitterionic monomer and PEGDA can render the resulting crosslinked polymer matrix superhydrophilic, and thus less likely to cause a foreign body response (FBR) (see U.S. Provisional Patent Application No. 63 / 192552, the contents of which are hereby expressly incorporated by reference in their entirety).

[0113] In some embodiments, the hydrogel material may be crosslinked via click chemistry. For example, copolymers comprising zwitterionic monomers and aldehyde motifs (e.g., [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (DMAPS)-aldehyde, referred to herein as "DMAPS-Ald"), as well as zwitterionic monomers and hydrazide motifs (e.g., DMAPS-hydrazide, referred to herein as "DMAPS-Hzd") may be used (see U.S. Provisional Patent Application No. 63 / 192552). The aldehyde readily reacts with the hydrazide to form a covalently crosslinked hydrogel. Due to the presence of zwitterionic monomers in the polymer backbone, these polymers may exhibit low protein binding properties. In an embodiment, one of these polymers may be blended with alginate within the shell. After printing, when this structure is immersed in a solution containing the opposing component, crosslinking occurs by covalent bonds between the fibers, resulting in F-F bonds.

[0114] In embodiments, the input material comprises microparticles, and as used herein, "microparticles" generally refers to miscible particles in the range of about 0.1 μm to about 100 μm, typically composed of polymers, metals, or other inorganic materials. They may be symmetric (e.g., spherical, cubic, etc.), but this is not a requirement. Microparticles having an aspect ratio of 2:1 or greater may be considered micro-rods or micro-fibers.

[0115] The input materials according to the embodiments of this specification can include any of a variety of natural or synthetic polymers that support the viability of living cells, such as, for example, alginate, laminin, fibrin, hyaluronic acid, poly(ethylene) glycol-based gels, gelatin, chitosan, agarose, or combinations thereof. In some embodiments, the subject bioink composition is physiologically compatible, that is, it promotes cell growth, cell differentiation, and intercellular communication. In certain embodiments, the input material includes one or more physiological matrix materials, or combinations thereof. "Physiological matrix material" means a biologically derived substance found in natural mammalian tissues. Non-limiting examples of such physiological matrix materials include fibronectin, thrombospondin, glycosaminoglycans (GAGs) (such as hyaluronic acid, chondroitin-6-sulfate, dermatan sulfate, chondroitin-4-sulfate, or keratan sulfate), deoxyribonucleic acid (DNA), adhesive glycoproteins, and collagen (such as collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, or collagen XVIII).

[0116] Collagen provides tensile strength to most tissues, and multiple collagen fibrils with a diameter of about 100 nm bind together to form strong multi-coil fibers with a diameter of about 10 μm. The biomechanical function of certain tissue constructs is provided in an oriented manner through the alignment of collagen fibers. In some embodiments, the input material includes collagen fibrils. Using an input material that includes collagen fibrils, a fibrous structure can be created that is formed into a tissue construct. By modifying the diameter of the fibrils, the collagen fibrils can be controlled to induce the polymerization of the collagen fibrils in a desired manner.

[0117] For example, previous studies have shown that microfluidic channels of different diameters can induce the polymerization of collagen fibrils along the length of the channels, but can form oriented fibrils only at channel diameters of 100 μM or less (Lee et al., 2006). Primary endothelial cells grown on these oriented matrices have been shown to align in the direction of the collagen fibrils. In another study, Martinez et al. demonstrated that 500 μm channels within a cellulose bead scaffold can induce the alignment of collagen and cells (Martinez et al., 2012). By adjusting the diameter of the fibrils, the orientation of the collagen fibrils within the fibrillar structure can be controlled. Thus, the fibrillar structure and the collagen fibrils therein can be patterned to generate a tissue construct having the desired configuration of collagen fibrils that is essential for imparting the desired biomechanical properties to the 3D printed structure.

[0118] Additional fluid Aspects of the present invention include one or more buffers. Buffers according to embodiments of the present invention are miscible with input materials (e.g., hydrogels) and do not crosslink the input materials. In some embodiments, the buffer includes an aqueous solvent. Non-limiting examples of buffers include polyvinyl alcohol, water, glycerol, propylene glycol, sucrose, gelatin, or any combination thereof.

[0119] Buffers according to embodiments of the present invention can have a viscosity in the range of about 1 mPa·s to about 5,000 mPa·s, such as a viscosity of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,250, 3,500, 3,750, 4,000, 4,250, 4,500, or 4,750 mPa·s. In some embodiments, the viscosity of the buffer can be adjusted to match the viscosity of one or more input materials.

[0120] Aspects of the present invention include one or more sheath fluids. A sheath fluid according to an embodiment of the present invention is a fluid that can be used to at least partially encapsulate or "sheath" an input material dispensed from a dispensing channel. In some embodiments, the sheath fluid includes an aqueous solvent. Non-limiting examples of sheath fluids include polyvinyl alcohol, water, glycerol, propylene glycol, sucrose, gelatin, or any combination thereof. A sheath fluid according to an embodiment of the present invention has a viscosity in the range of about 1 mPa·s to about 5,000 mPa·s, for example, a viscosity of 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.

[0121] In some embodiments, the sheath fluid includes a chemical cross-linking agent. In some embodiments, the chemical cross-linking agent includes a divalent cation. Non-limiting examples of divalent cations include Cd 2+ 、Ba 2+ 、Cu 2+ 、Ca 2+ 、Ni 2+ 、Co 2+ 、or Mn 2+ 。In a preferred embodiment, Ca 2+ is used as the divalent cation. In some embodiments, the concentration of divalent cations in the sheath fluid is in the range of about 80 mM to about 140 mM, for example, about 90, 100, 110, 120, or 130 mM.

[0122] Cell population In an embodiment, the cell population is selected from the group consisting of or including a single cell suspension, cell aggregates, cell spheroids, cell organoids, or combinations thereof. The input material according to an embodiment of the present invention can include, but is not limited to, any mammalian cell type including stem cells (e.g., embryonic stem cells, adult stem cells, induced pluripotent stem cells), germ cells, endodermal cells (e.g., lung cells, liver cells, pancreatic cells, gastrointestinal tract cells, or urogenital tract cells), mesodermal cells (e.g., kidney cells, bone cells, muscle cells, endothelial cells, or heart cells), ectodermal cells (skin cells, nervous system cells, pituitary cells, or eye cells), stem cell-derived cells, or any combination thereof.

[0123] For example, the input material includes cells derived from endocrine glands and exocrine glands including pancreas (α, β, δ, ε, γ), liver (hepatocytes, Kupffer cells, stellate cells, sinusoidal cells), thyroid (follicular cells), pineal gland (pinealocytes), pituitary gland (growth hormone-producing cells, mammotropic hormone-secreting cells, gonadotropic hormone-secreting cells, adrenocorticotropic hormone-secreting cells, and thyroid-stimulating hormone-producing cells), thymus (thymocytes, thymic epithelial cells, thymic stromal cells), adrenal gland (cortical cells, chromaffin cells), ovary (granulosa cells), testis (Leydig cells), gastrointestinal tract (enteroendocrine cells - intestine, stomach, pancreas), fibroblasts, chondrocytes, meniscus fibrocartilage cells, bone marrow stromal (stem) cells, embryonic stem cells, mesenchymal stem cells, induced pluripotent stem cells, differentiated stem cells, tissue-derived cells, smooth muscle cells, skeletal muscle cells, cardiomyocytes, epithelial cells, endothelial cells, myoblasts, chondroblasts, osteoblasts, osteoclasts, and any combination thereof.

[0124] Cells can be obtained from a donor (allogeneic), from a species different from the recipient (xenogeneic), or from the recipient (autologous). Specifically, in embodiments, cells can be obtained from a suitable donor such as a human or an animal, or from the subject into which the cells are to be transplanted. Mammalian species include, but are not limited to, humans, monkeys, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, and rats. In one embodiment, the cells are human cells. In other embodiments, the cells can be derived from an animal such as a dog, cat, horse, monkey, or any other mammal.

[0125] In some embodiments, the at least one biological substance comprises a cell population that expresses / secretes one or more endogenous bioactive substances (s), such as insulin, glucagon, ghrelin, pancreatic polypeptide, factor VII, antihemophilic factor, factor IX, alpha1-antitrypsin, angiogenic factors, growth factors, hormones, antibodies, enzymes, proteins, exosomes, and the like. The endogenous bioactive agents described herein include agents that cells naturally produce under biological circumstances (e.g., insulin release in response to elevated glucose concentration). Endogenous bioactive agents can constitute therapeutic agents with respect to the present disclosure.

[0126] In some embodiments, the input material can include genetically modified cells that secrete a specific factor. In embodiments, it is within the scope of the present disclosure that the above-described cell population can include modified cells (e.g., genetically modified cells) that secrete a specific factor. The cells can also be cells derived from an established cell culture line or cells that have undergone genetic modification and / or genetic manipulation to obtain a desired genotype or phenotype. In some embodiments, tissue pieces can also be used, which can provide several different cell types within the same structure.

[0127] Gene modification techniques applicable to the present disclosure include recombinant DNA (rDNA) technology (Stryjewska et al., Pharmacologial Reports. 2013;65:1075), cell modification based on the use of targeted nucleases (e.g., meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR-associated nuclease Cas 9 (CRISPR-Cas 9 ) (Lim et al., Nature Communications. 2020;11:4043; Stoddard BL, Structure. 2011;19(1):7-15; Gaj et al., Trends Biotechnol. 2013;31(7):397-405; Hsu et al., Cell. 2014;157(6):1262; Miller et al., Nat Biotechnol. 2010;29(2):143-148), cell modification based on the use of site-specific recombination using recombinase systems (e.g., Cre-Lox) (Osborn et al., Mol Ther. 2013;21(6):1151-1159; Hockemeyer et al., Nat Biotechnol. 2009;27(9):851-857; Uhde-Stone et al., RNA. 2014;20(6):948-955; Ho et al., Nucleic Acids Res. 2015;43(3):e17; Sengupta et al., Journal of Biological Engineering. 2017;11(45):1-9), etc., but are not limited thereto. In some embodiments, some combinations of the above-described techniques may be used for cell modification.

[0128] The present disclosure encompasses recombinant cells capable of producing one or more therapeutic agents, including but not limited to proteins, peptides, nucleic acids (e.g., DNA, RNA, mRNA, siRNA, miRNA, nucleic acid analogs), peptide nucleic acids, aptamers, antibodies or fragments or portions thereof, antigens or epitopes, hormones, hormone antagonists, growth factors or recombinant growth factors and fragments and variants thereof, cytokines, enzymes, antibiotics or antibacterial compounds, anti-inflammatory agents, antifungal agents, antiviral agents, toxins, prodrugs, small molecules, drugs (e.g., pharmaceuticals, dyes, amino acids, vitamins, antioxidants), or any combination thereof.

[0129] In embodiments, the cells of the present disclosure may be modified to include at least one mechanism for providing local immunosuppression at the site of transplantation when transplanted into an allogeneic host, e.g., in the tissue fibers of the present disclosure. In examples, the cells may include a series of transgenes, each transgene being cytoplasmic, membrane-bound, or locally acting, the function of which improves the activation and function of antigen-presenting cells, improves the activity or cytolytic function of leukocytes that attack the graft, improves the macrophage cytolytic function and phagocytosis of the transplanted cells, induces apoptosis in leukocytes that attack the graft, improves local inflammatory proteins, and protects from leukocyte-mediated apoptosis, and may encode gene products including but not limited to these (WO2018 / 227286; Harding et al., BioRxiv. 2019; DOI: 10.1101 / 716571; Lanza et al., Nature Reviews Immunology. 2019; 19:723-733l; Harding et al., Cell Stem Cell. 2020; 27(2):198-199).

[0130] In embodiments, the cells of the present disclosure can be modified by a method for controlling cell proliferation. As an example, the cells may be genetically modified at a cell division locus (CDL) to include a negative selectable marker and / or an inducible activator-based gene expression system, thereby controlling the allowing, removal, and / or suppression of the growth of the genetically modified cells by the addition or removal of an appropriate inducer (WO2016 / 141480; Liang et al., Nature. 2018; 563(7733): 701-704).

[0131] Suitable growth conditions for mammalian cells are well known in the art (Freshney, R.I. (2000) Culture of Animal Cells, a Manual of Basic Technique. Hoboken N.J., John Wiley & Sons; Lanza et al. Principles of Tissue Engineering, Academic Press; 2nd edition, May 15, 2000; and Lanza & Atala, Methods of Tissue Engineering Academic Press; 1st edition, October 2001). Cell culture media generally contain essential nutrients and, optionally, additional elements such as growth factors, salts, minerals, vitamins, etc. that can be selected according to the cell type(s) to be cultured. Specific components may be selected to improve cell growth, differentiation, specific protein secretion, etc. Generally, standard growth media contain 110 mg / L of pyruvate and glutamine, and are supplemented with 10-20% fetal bovine serum (FBS) or calf serum, and low-glucose Dulbecco's modified Eagle's medium (DMEM). 100 U / ml of penicillin is suitable as well as various other standard media well known in the art. Growth conditions vary depending on the mammalian cell type used and the desired tissue.

[0132] In some embodiments, cell type-specific reagents can be advantageously utilized in the subject input materials for use with the corresponding cell types. For example, an extracellular matrix (“ECM”) can be directly extracted from a subject's tissue and then solubilized and incorporated into the input material to generate a tissue-specific input material for the tissue to be printed. Such ECMs can be readily obtained from a patient's sample and / or from suppliers such as zPredicta (rBone™, available at zpredicta.com / home / products).

[0133] Printing system Bio-printing systems vary, but generally a bio-printing system includes at least one reservoir containing an input material (e.g., bioink, sheath fluid, buffer, etc.) for dispensing through a dispensing orifice (e.g., a dispensing orifice associated with a needle, nested needle, syringe, nozzle, etc.). The dispensing orifice(s) can include suitable shape(s), such as, for example, circular, square, elliptical, oblong, rectangular, etc. In some examples, a bio-printing system can include a plurality of reservoirs and / or means for selecting a reservoir for use in bio-printing from the plurality of reservoirs. A bio-printing system including one or more reservoirs can be advantageous in continuous or substantially continuous bio-printing applications. The volume of the reservoir can vary, for example, between about 100 pl and about 1 L or more, including any intervening value therein, such as, for example, 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, for example, between about 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or 950 mL.

[0134] In embodiments, the reservoir may be made of an inner material that resists cell attachment thereto. In embodiments, at least the inner side of the reservoir may be made of a biocompatible material. The reservoir may be compatible with bioprinting involving extruding a semi-solid or solid bioink, or support material, through one or more dispensing orifices. The reservoir may be compatible with bioprinting involving dispensing a liquid or semi-solid cell solution, cell suspension, or cell concentrate through one or more dispensing orifices. The reservoir may be compatible with discontinuous bioprinting, continuous bioprinting, and / or substantially continuous bioprinting. The reservoir may include, but is not limited to, capillaries, micropipettes, syringes, needles, bottles, washbasins, receptacles, etc. Many inner diameters are suitable for substantially circular or cylindrical reservoirs.

[0135] In some examples, the reservoir of the bioprinting system may be primed. For example, priming the reservoir can improve the accuracy of the dispensing process and / or the volumetric process, such as by compressing and advancing the contents of the reservoir until the dispensed material (e.g., bioink) is positioned at a location in contact with the dispensing orifice.

[0136] In a preferred embodiment, the bioprinting system includes the technologies described in WO2014 / 197999, WO2018 / 165761, WO2020 / 056517, WO2021 / 081672, and U.S. Provisional Patent Application No. 63 / 290595, the disclosures of which are hereby expressly incorporated by reference herein. As detailed therein, the disclosed bioprinting system and its components enable switching between multiple materials, so that the composition of one or more components of the synthetically generated tissue fibers (e.g., cell type, biomaterial composition) can be modified along the length of the fibers while continuously printing. In an embodiment, the microfluidics-based bioprinting system is an RX1™ bioprinter (Aspect Biosystems, Vancouver, BC, Canada).

[0137] In an exemplary embodiment of a preferred bioprinting system, the system includes a print head that includes a dispensing channel, and one or more material channels and a core channel converge at a proximal end of the dispensing channel. The print head can be configured to dispense a buffer and / or a sheath fluid simultaneously with one or more crosslinkable materials. In some embodiments, the print head is configured to maintain a constant mass flow rate through the dispensing channel. In this way, the print head can be configured to facilitate the smooth and continuous flow of one or more input materials (or a mixture of one or more input materials) and a buffer and / or a sheath fluid through the dispensing channel. As described in more detail in WO2020 / 056517, in the use of such a print head, the input material flowing through the dispensing channel can be crosslinked from the inside by the fluid flowing through the core channel and / or from the outside by the sheath fluid flowing through the downstream sheath fluid channel. In some embodiments, as described in detail in WO2021 / 081672 and U.S. Provisional Patent Application No. 63 / 290595, the print head includes one or more fluid focusing chambers having a frustoconical shape and optionally one or more print head adapters. In an embodiment, the print head is a DUO™ microfluidic print head, or a CENTRA™ microfluidic print head (Aspect Biosystems, Vancouver, BC, Canada).

[0138] Other examples of bioprinting systems relevant in the context of the present disclosure include, but are not limited to, 3-D Bioplotter® (EnvisionTEC Inc., Dearborn, MI, USA), NovoGen Bioprint® platform (Organovo®, San Diego, CA, USA), R-Gen100 and R-Gen200 (RegenHU, Viras-Saint-Pierre, Switzerland), Bioprinter Fabion and Fabion 2 (3D Bioprinting Solutions, Moscow, Rusia), BioBot® Basic, BioAssemblyBot® 200 / 400 / 500 (Advanced Solutions, Louisville, KY, USA), BIO X™, BIO X6™, INKREDIBLE+™ (CellINK, Boston, MA, USA), Ourobotics Revolution (Ourbotics, Cork, Ireland), BioScaffolder2.1 (GeSim, Radeberg, Germany), Omega Bioprinter (3Dynamic Systems, Bridgend, UK), Syn^ and Explorer (Bio3D, Singapore), Alevi 1 / 2 / 3 (Alevi by 3D Systems, Rock Hill, SC, USA), and Dr.Invivo 4D6 (Rokit Healthcare, Seoul, Korea).

[0139] As described above, a bioprinting system typically dispenses a bioprinted material onto a receiving surface. The bioprinted fibrous structures described herein are printed using means for suspending the bioprinted fibrous structure during one or more of printing, patterning, and / or processing. In embodiments, the fibrous structure can be printed onto a receiving surface (e.g., mesh 514 in FIGS. 5A - 5B) by using means for suspending the structure (e.g., frame 508 in FIGS. 5A - 5B). In embodiments, since the fibrous structure can be printed by means for suspending the structure disclosed herein, the fibrous structure remains fully suspended away from the receiving surface during printing.

[0140] The bioprinting system disclosed herein can be modified or otherwise adapted to provide a surface that can correspond to means for suspending a bioprinted tissue structure disclosed herein. In embodiments, the surface can be adapted or otherwise configured in a manner that operably couples to one or more containers (e.g., containers, multi - well plates, etc.). For example, without limitation, the containers can include containers that can hold solutions such as cross - linker solutions or immersion coating solutions described herein. In embodiments, such containers can be sized and shaped to accommodate the entire means for suspending the bioprinted fibers therein, and the liquid held within the container can fully immerse the means for suspending the bioprinted fibers and then fully immerse the bioprinted fibrous structure attached thereto.

[0141] In embodiments, the receiving surface is disposable. In embodiments, the receiving surface can be effectively sterilized. In embodiments, the receiving surface includes solids, semi - solids, or some combination thereof. In embodiments, the receiving surface is porous. In embodiments, the receiving surface includes glass, coated glass, plastic, coated plastic, metal, metal alloy, mesh, lattice, or a combination thereof.

[0142] In some embodiments, the bioprinting system includes a fluid removal component for removing excess fluid (e.g., excess sheath fluid and / or excess buffer) from the receiving surface and / or from the surface of the dispensed tissue fiber structure. During printing, excess fluid may potentially accumulate or "pool" on the receiving surface or on the surface of the dispensed tissue fiber structure, and in some instances, such pooling may interfere with one or more aspects of the deposition process. For example, in the context of the present disclosure, unwanted excess fluid may potentially reduce the ability of the fiber structure to adhere to the struts (e.g., struts 206 of FIG. 2A) of the means (e.g., frame 202 of FIG. 2) for suspending the bioprint fiber structure, and may add an unwanted weight (possibly unevenly distributed) to the bioprint fibers and the like, such that the dispensed fibers may deviate from their intended positions within the printed 3D structure. Thus, in some embodiments, the three-dimensional structure fabrication may be improved by removing excess sheath fluid from the receiving surface and / or from the surface of the dispensed fiber structure by the fluid removal component.

[0143] Excess fluid may be removed from the receiving surface or from the surface of one or more layers of the dispensed fibers by withdrawing the fluid from these surfaces, by enabling or facilitating evaporation of the fluid from these surfaces, or, in embodiments where the receiving surface is porous, excess fluid may be removed by withdrawing it through the porous surface. In some embodiments, an absorbent material (e.g., a sponge) may be used to pull excess fluid away from the receiving surface.

[0144] In some embodiments, the receiving surface includes a vacuum component (e.g., vacuum chuck 512 of FIGS. 5A-5B) configured to apply suction to the receiving surface from one or more vacuum sources. 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 component is configured to suction excess fluid from the receiving surface before, during, and / or after execution of the printing process. In some embodiments where the receiving surface is porous, the vacuum component may be configured to apply suction to draw excess fluid through the porous surface.

[0145] In some embodiments, the receiving surface includes one or more tubes fluidly connected to a vacuum source, and the vacuum source can provide suction for removing excess fluid from the receiving surface and optionally from the surface of the dispensed fibrous structure. In such embodiments, a solid or porous receiving surface can also be used. In some embodiments, the printhead (e.g., a microfluidics-based printhead) is further configured to include one or more vacuum channels, each of which has an orifice located near (i.e., adjacent to) the dispensing orifice. When the printhead is in fluid communication with the vacuum, the one or more vacuum channels can direct a negative pressure to the area of the receiving surface where material has been dispensed from and / or into a portion of the surface area of the dispensed fibrous structure, thereby pulling up excess fluid from the receiving surface and / or from the surface of the dispensed fibrous structure.

[0146] In some embodiments, the bioprinter system may use dispensing means to control the flow of material in fiber generation. For example, the bioprinter system may use displacement of the material to control the flow of the material. In an embodiment, the dispensing means provides a force to dispense one or more materials to be dispensed. In one embodiment, the dispensing means provides pneumatic pressure to supply a force for dispensing one or more materials. In some embodiments, the bioprinter system may be composed of one or more pumps. Examples include, but are not limited to, gear pumps, peristaltic pumps, lobe pumps, piston pumps (e.g., dual piston pumps), syringe pumps, and the like. In some embodiments, the pump applies a force to the material contained in the reservoir to assist in moving the material toward an associated dispensing orifice.

[0147] In a preferred example, the bioprinter system used in the context of the present disclosure includes a radial pump assembly configured to minimize the internal volume within a multi-channel bioprinting system, preferably a microfluidic bioprinting system, including a plurality of pumps positioned in a radial array on a mounting bracket, each of the plurality of pumps including a housing and a retainer for fixing the pump. A related example of such a system is described in detail in U.S. Provisional Patent Application No. 63 / 290595, the content of which is hereby incorporated by reference in its entirety.

[0148] In an embodiment, a bioprinting system related to the present disclosure can include some type of enclosure system. The enclosure system can be configured to enclose part or all of the bioprinting system. Preferably, the enclosure system encloses at least one region where bioprinting fibers are dispensed, such as, for example, at least one receiving surface, preferably at least one receiving surface and one or more print heads, and optionally encloses any dispensing means (e.g., syringe pump(s)) configured to deliver a flow material to a dispensing orifice, and optionally encloses one or more reservoirs. In an embodiment, such an enclosure system can be configured to adopt an open configuration that allows access to aspects of the bioprinting system, and a closed configuration in which certain aspects of the bioprinting system are sealed or substantially sealed from the ambient atmosphere.

[0149] By relying on the enclosure system, precise control over parameters including, but not limited to, temperature, humidity, O 2 , and CO 2 can be achieved. By precisely controlling such parameters, it may be possible to enhance the stability and integrity of the 3D bioprinted structure, as well as increase the survival of cells during and after the bioprinting process under conditions where the 3D bioprinted structure contains cellular material. In an embodiment, temperature, humidity, O 2 , and CO 2Variables such as can be controlled within the enclosure system via a feedback control system (e.g., a proportional-integral-derivative (PID) controller). See, for example, Matamoros M, et al. (2020) Micromachines, 11, 999. In embodiments, such a feedback control system can, for example, stabilize the temperature within the enclosure system to a desired temperature (within some range of error) and / or stabilize the humidity within the enclosure system to a desired humidity (within some range of error). In some additional or alternative embodiments, such a feedback control system can 2 stabilize CO to a desired level (e.g., a desired ppm within some range of error). In some additional or alternative embodiments, such a feedback control system can 2 stabilize O to a desired level (e.g., a desired ppm within some range of error).

[0150] Thus, in embodiments, the bioprinting system related to the present disclosure can include one or more of a temperature regulation component, a humidity regulation component, an O 2 regulation component, and a CO 2 regulation component. In some embodiments, the temperature regulation component can include a heater (e.g., a radiant heater, a convective heater, a conductive heater, a fan heater, a heat exchanger, or any combination thereof). In embodiments, the temperature regulation component can include a cooling element (e.g., a coolant, a cooling liquid, a Peltier cooler, a radiative cooler, a convective cooler, a conductive cooler, a fan cooler, or any combination thereof). The humidity regulation component can include, for example, a chamber (e.g., a tank) of water that can be evaporated by a piezoelectric transducer. Control of O 2 can be by O 2 injection. Control of CO 2 can be by CO 2It may be by injection. In some embodiments, the temperature regulating component can adjust and / or maintain the temperature in one or more of the enclosure system and / or the print head, printer stage, receiving surface, input material, and / or fluid, flow material, and / or fluid (e.g., sheath fluid and / or buffer fluid). In an embodiment, the temperature regulating component is configured to adjust the temperature to a set point in the range of 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. In an embodiment, the humidity regulating component is configured to adjust the humidity between about 30% and 100%, such as about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%. In an embodiment, the CO 2 regulating component is configured to adjust the CO 2 level to be between about 2% and about 15%, such as about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%.

[0151] In an embodiment, the 3D printing system related to the present disclosure achieves a specific geometry of the dispensed fiber structure by moving a printer stage or surface (e.g., receiving surface) having means for suspending the 3D printed fiber structure located thereon relative to the dispensing orifice. In an alternative embodiment, the 3D printing system related to the present disclosure achieves a specific geometry of the dispensed fiber structure by moving the dispensing orifice (optionally, a plurality thereof) relative to a printer stage or surface (e.g., receiving surface) having means for suspending the 3D printed fiber structure located thereon or above. In a particular embodiment, at least a portion of the 3D printing system is maintained in a sterile environment (e.g., within a biosafety cabinet (BSC)). In some embodiments, the 3D printing system is configured to be compatible with a completely sterile environment.

[0152] In some embodiments, the bioprinting system includes a 3D motorized stage, also referred to herein as a positioning unit, that includes at least three arms for positioning a surface (e.g., a receiving surface) having means for suspending a bioprinted fibrous structure located thereon in a three-dimensional space below the dispensing orifice (i.e., along the x, y, and z axes of a Cartesian coordinate system). In some additional or alternative embodiments, a similar positioning unit positions the dispensing orifice in a three-dimensional space above a surface (e.g., a receiving surface) having a system for suspending a bioprinted fibrous structure located thereon or above it.

[0153] In some embodiments, the arms of the 3D motorized stage are each driven by three corresponding motors and controlled by a programmable control processor such as a computer. In one embodiment, a surface (e.g., a receiving surface) having means for suspending a bioprinted fibrous structure located thereon is movable along all three principal axes of a Cartesian coordinate system by the 3D motorized stage, and the movement of the stage is defined by using computer software. In some additional or alternative embodiments, the dispensing orifice (or orifices) is movable along all three principal axes of a Cartesian coordinate axis by the 3D motorized stage, and the movement of the stage is defined using computer software.

[0154] It should be understood that the present invention is not limited only to the positioning system described, and that external tail positioning systems are known in the art. When the material is dispensed from a dispensing orifice (e.g., on a microfluidic printhead), the positioning unit moves in a pattern controlled by software, thereby creating a first layer of bioprint fibers to be suspended around a support (e.g., support 206 in FIG. 2A) of a means (e.g., frame 202 in FIG. 2A) for suspending the bioprint fibers. Next, additional layers of the dispensed material are stacked, and as a result, the final 3D geometry of the layers of the dispensed material is typically a replication of the 3D geometry design provided by the software. The 3D design can be created using common 3D CAD (computer-aided design) software, as is known in the art, or can be generated from a digital image. Further, according to one embodiment of the present invention, if the geometry generated by the software includes information regarding the specific material used, it is possible to assign a particular type of flow material to different geometric locations. For example, in some embodiments, the 3D structure to be printed can include two or more different input materials, each input material having different properties (e.g., each input material includes different cell types, different cell concentrations, different extracellular matrix (ECM) compositions, different types of materials (e.g., different types of hydrogel materials), etc.).

[0155] In some embodiments, the bioprinting system includes, optionally, a light module for exposing a photo-crosslinkable flow material to light in order to crosslink the material. The light module (e.g., an ultraviolet (UV) light module) can be integrated within the bioprinting system (e.g., within its printhead) in some embodiments, or can be a stand-alone component of such a system in other embodiments. In some embodiments, the light module is ring-shaped. In some embodiments, the ring-shaped light module completely surrounds a transparent portion of the dispensing channel (or dispensing needle / nozzle / syringe) and / or the dispensing orifice. In some embodiments, the ring-shaped light module is positioned immediately below the dispensing orifice.

[0156] In an embodiment, the ring-shaped module can be composed of a plurality of light sources so as to direct light inwardly in the direction of the central cavity of the ring-shaped optical module. As a result, as the fibers are printed, the light is directed circumferentially by the fibers. In an embodiment, the plurality of light sources includes individual light sources between at least 10 and 40, for example, between 15 and 35, for example, between 20 and 30. In an embodiment, the light source includes a light emitting diode (LED), such as a UV-LED. Related examples of such ring-shaped light sources and their incorporation into the bioprinting system are described in detail in U.S. Provisional Patent Application No. 63 / 290595, the contents of which are hereby incorporated by reference in their entirety.

[0157] Aspects of the bioprinting system include a software program, which is configured to facilitate deposition of a subject flow material in a particular pattern and at a particular location to form a particular planar or 3D structure in a manner where the structure is deposited around struts (e.g., strut 206 in FIG. 2A) of means (e.g., frame 202 in FIG. 2A) for suspending bioprint fibers as described herein. To create such a structure, the subject printing system weaves continuous bioprint fibers in a pattern defined around at least two, optionally more than two, optionally more than ten, optionally more than fifteen, optionally more than twenty, optionally more than twenty-five, optionally more than thirty, optionally more than thirty-five, optionally more than forty, optionally more than forty-five, optionally more than fifty, optionally more than seventy-five, optionally more than one hundred struts (e.g., strut 206 in FIG. 2), which are part of means (e.g., frame 202 in FIG. 2) for suspending the bioprint fiber structure and / or a manufacturing platform (e.g., manufacturing platform 500 in FIGS. 5A - 5B), to deposit the subject input material in an accurate (two - dimensional or three - dimensional) manner. The resulting structure can include 1, 2, 3, 4, 5, or more than 5 layers, such as 10 or more layers, 20 or more layers, 30 or more layers, 40 or more layers, 50 or more layers, 60 or more layers, 70 or more layers, 80 or more layers, 90 or more layers, 100 or more layers, 110 or more layers, 120 or more layers, 130 or more layers, 140 or more layers, 150 or more layers, 160 or more layers, 170 or more layers, 180 or more layers, 190 or more layers, 200 or more layers. In some embodiments, the method by which the printing system deposits the material (i.e., the location and overall deposition pattern) is defined by user input and converted into computer code. In some embodiments, the computer code includes a series of instructions executable on a central processing unit (CPU) of a digital processing device created to perform a particular task. In some embodiments, print parameters including, but not limited to, dimensions of the printed fibers, pump speed, movement speed of the positioning system, and strength or concentration of the cross - linking agent are defined by user input and converted into computer code.In some embodiments, rather than being directly defined by user input, the printing parameters are derived from other parameters and conditions by computer code.

[0158] Aspects of the present invention include a method for creating a fibrous structure, the method including a computer module receiving an input of a visual representation of a desired tissue construct, the computer module generating a series of commands, the commands being based on the visual representation and being readable by a bioprinting system as disclosed herein, the computer module providing the series of commands to the bioprinting system, and the printing system depositing one or more input materials according to the commands to form a fibrous structure having a defined geometry.

[0159] In some embodiments, the method by which the bioprinting system deposits the input material (i.e., the position and overall deposition pattern) is defined by user input and converted into computer code. In some embodiments, the devices, systems, and methods disclosed herein further include a non-transitory computer-readable storage medium or memory medium encoded with computer-readable program code. In some embodiments, the computer-readable storage medium is a tangible component of a computer connected to the bioprinting system (or a component thereof) or the bioprinting system (or a component thereof). In some embodiments, the computer-readable storage medium is optionally removable from a digital processing device. In some embodiments, examples of the computer-readable storage medium include, but are not limited to, CD-ROMs, DVDs, flash memory devices, solid state memories, magnetic disk drives, magnetic tape drives, optical disk drives, as well as cloud computing systems and / or services. In some cases, the programs and instructions are encoded permanently, almost permanently, semi-permanently, or non-transitorily on the storage medium.

[0160] In some embodiments, the devices, systems, and methods described herein include software, servers, and database modules. In some embodiments, a "computer module" is a software component (including code sections) that interacts with a larger computing system. In some embodiments, a software module (or program module) is provided in the form of one or more files and typically processes a specific task within a computing system.

[0161] In some embodiments, a module is included in one or more software systems. In some embodiments, a module is integrated with one or more other modules to form one or more software systems. A computer module can optionally be a stand-alone code section or optionally be code that cannot be individually identified. In some embodiments, a module exists within a single application. In another embodiment, a module exists within multiple applications. In some embodiments, a module is hosted on one machine. In some embodiments, a module is hosted on multiple machines. In some embodiments, a module is hosted on multiple machines in one location. In some embodiments, a module is hosted on multiple machines in multiple locations. By the computer modules according to the embodiments of the present invention, an end user can use a computer to execute one or more aspects of the methods described herein.

[0162] In some embodiments, the computer module comprises a graphical user interface (GUI). As used herein, "graphical user interface" means a user environment that uses graphical and textual representations of application inputs and outputs, as well as hierarchical or other data structures in which information is stored. In some embodiments, the computer module comprises a display screen. In further embodiments, the computer module presents a two-dimensional GUI via the display screen. In some embodiments, the computer module presents a three-dimensional GUI, such as a virtual reality environment, via the display screen. In some embodiments, the display screen is a touch screen and presents an interactive GUI.

[0163] Quality management system For example, quality assurance of 3D bioprinting of fibers by a printing system, such as the printing system disclosed herein, is essential for the production of reproducible biofibers, their functionality, and regulatory approval for any conversion applications. Accordingly, the disclosed bioprinting system can incorporate one or more of the quality management systems described below. In an embodiment, the quality management system includes one or more cameras. In embodiments where at least a portion of the dispensing orifice (and / or dispensing channel / needle / syringe / nozzle following it), and / or other aspects of the system, such as a microfluidic print head, are transparent, one or more cameras can be used to image the flow of the dispensed material for the purpose of detecting blockages or other abnormalities in the flow of the material.

[0164] For example, in embodiments, the microfluidic printhead of the present disclosure (e.g., similar or substantially the same as the DUO™ microfluidic printhead or the CENTRA™ microfluidic printhead (Aspect Biosystems, Vancouver, BC, Canada)) includes a transparent dispensing channel. In such embodiments, the camera system can include a first camera disposed at a first angle with respect to the transparent dispensing channel and a second camera disposed at a second different angle with respect to the transparent dispensing channel. In embodiments, the two cameras may be oriented at an angle of about 90° with respect to each other. The first camera and the second camera can form part of a machine learning-based system that can identify one or more deviations of the material flow from the parameters of the material flow established by the user. The method. For example, such a system may enable monitoring of fiber concentricity, various fiber properties, clogging, presence or absence of bubbles, etc., and further control one or more parameters (e.g., opening and closing of valves, material flow rate, etc.) based on such monitoring. This concept and its variations are described in U.S. Provisional Application No. 63 / 238028, the content of which is hereby incorporated by reference in its entirety.

[0165] In embodiments, one or more additional or alternative cameras can be included as part of the bioprinting system of the present disclosure. In such an embodiment, the camera(s) can be aimed at means for suspending the bioprinted fiber structure (e.g., frame 202 of FIG. 2) so as to image the bioprinted fiber structure when it is printed and enable monitoring of one or more properties associated with the fibers during printing. Such properties include, but are not limited to, the presence or absence of leading and / or trailing fibers and the fidelity of the shape of the printed fibers and / or the 3D structure formed from the printed fibers.

[0166] In an embodiment, the ring-shaped optical module such as the above-described ring-shaped optical module may form part of a quality control system, and in that regard, the ring-shaped optical module may help ensure the uniformity of crosslinking when fibers made of a photocrosslinkable material are printed.

[0167] Also, in this specification, it is recognized that the use of pneumatic valves may be advantageous in the context of the present disclosure in reducing or avoiding inaccuracies or distortions in a desired 3D structure. For example, depending on one or more pneumatic valves in conjunction with the printing system of the present disclosure, it may reduce or avoid inaccurate start and / or stop of the printing process that may otherwise lead to the formation of precursor fibers and / or subsequent fibers that distort the dimensions of the printed fibers and the 3D structure. Also, depending on pneumatic valves may be advantageous in accurately switching the type of material from one to the other without forming a tip and / or a terminus, in fibers composed of multiple types of materials along the length of the fiber. Such accurate switching can enable effective compartmentalization of different segments along the length of the fiber. For example, the fiber may include a first segment made of a first type of hydrogel and a second segment made of a second type of hydrogel, and the second segment may optionally include cells and a third segment made of yet another type of hydrogel. Such examples are intended to be illustrative, and one of ordinary skill in the art can select the type of material and optionally the cell type(s) (or the absence thereof) for various compartments along the length of the fiber as desired for a particular application.

[0168] The above-described embodiments and examples are merely illustrative and are intended to be non-limiting. One of ordinary skill in the art will be able to recognize or confirm numerous equivalents of specific compounds, materials, and procedures using only routine experimentation. All such equivalents are considered to be within the scope of the present invention and are encompassed by the appended claims.

[0169] Manufacturing method Aspects of the present invention include methods of printing a linear fiber structure, a planar structure including one or more fiber structures, or a three-dimensional (3D) structure including two or more layers of a planar structure. Methods of printing a first layer and optionally a second layer, a third layer, etc. (e.g., 200 or more layers are within the scope of the present disclosure) are described in detail above with reference to FIGS. 3-4 and FIG. 6. In some embodiments, a linear fiber structure can be created by wrapping a bioprint fiber around a first strut (e.g., strut 206 in FIG. 2A) one or more times and then continuing to print the fiber and wrapping the fiber around a second strut one or more times, preferably where the first strut and the second strut are on opposite sides of a means for suspending the bioprint fiber structure (e.g., frame 202 in FIG. 2A). In some embodiments, the method first includes providing a design of the linear structure, planar structure, or 3D structure to be printed. The design can be created using commercially available CAD software. In some embodiments, the design includes information regarding a particular material to be assigned to a particular location within the structure(s) to be printed (e.g., in the case of a heterogeneous structure including multiple materials).

[0170] In an embodiment, the method includes dispensing bioprinted fibers into a crosslinking solution bath, where the fibers are printed via means for suspending the bioprinted fibers (e.g., frame 202 of FIG. 2), and then the obtained fibers are immersed in a crosslinking agent solution bath. In some embodiments of such a method, the bioprinted fibers may be further crosslinked before being dispensed into the crosslinking solution bath. For example, the bioprinted fibers can be crosslinked via a sheath fluid containing a crosslinking agent or by photocrosslinking before being introduced into the crosslinking agent solution bath. In other embodiments, the bioprinted fibers may not be crosslinked before being dispensed into the crosslinking solution bath. In still other embodiments, the bioprinted fibers are crosslinked (e.g., exposed to a sheath fluid containing a crosslinking agent and / or photocrosslinked) under conditions where the means for suspending the bioprinted fibers is not positioned within the crosslinking agent solution bath before being dispensed onto the means for suspending the bioprinted fibers. In some embodiments, after printing the crosslinked fibers, the means for suspension and the correspondingly attached bioprinted fibers can then be immersed in a crosslinking solution bath. Such embodiments have been described above with respect to the exemplary process flow of FIG. 8.

[0171] In an embodiment, the method includes uniformly coating a bioprinted fiber structure to produce a conformal coating over the entire outer surface of the structure. In an embodiment, the method includes coating bioprinted fibers (e.g., a 3D structure) printed using the means for suspension disclosed herein (e.g., frame 202 of FIG. 2A), and applying a desired coating material while the fiber structure is suspended above a receiving surface by the suspension means. In this way, a conformal coating containing the coating material can be added to the suspended bioprinted fibers. In an embodiment, the method includes producing a bioprinted fiber structure (e.g., a 3D structure) crosslinked by any of the methods described above, and then immersing the entire fiber structure in a coating solution while the fiber structure is attached to the means for suspending the bioprinted fiber structure.

[0172] In other additional or alternative embodiments, the coating solution remains attached to the means for suspending the fibrous structure and can be applied to the fibrous structure (e.g., by a print head such as a microfluidic print head) while the fibrous structure is suspended away from the receiving surface. As described above herein, in multiple embodiments, multiple (e.g., at least 2, 3, 4, 5, 6 or more) coating layers can be applied to the bioprint fibers.

[0173] In some embodiments of the methods described herein, the solution (e.g., a crosslinking agent solution, a buffer solution, a coating solution) in which the bioprint fibers are immersed can be removed by suction or by drainage (e.g., via one or more insertable orifices). In this way, a single container can be used to immerse the bioprint fibers in different solutions. For example, bioprint fibers attached to a suspending means (e.g., the frame 202 of FIG. 2A) can be immersed in a crosslinking agent solution held in a container and then the crosslinking agent solution can be suctioned or otherwise removed. Next, the container can be filled with another solution (e.g., a wash buffer, a coating solution, a preservation solution, etc.). In this way, the means for suspending the bioprint fibers and the attached fibers themselves do not need to be moved from one container to another during various processing steps. The rate of addition and / or removal of the solution can be controlled to minimize (e.g., not impart or substantially not impart) the disturbance imparted to the bioprint fibers during addition and / or removal.

[0174] In some embodiments of the methods described herein, to facilitate removal of one solution (e.g., a crosslinking agent solution) in which the bioprinted fibrous structure is immersed, and then optionally subsequent immersion of the fibrous structure into another solution (e.g., a coating solution) or other treatment (e.g., depositing a coating solution onto the fibrous structure), means for suspension (e.g., frame 202 of FIG. 2A) and lifting the entire adhered bioprinted fibers out of the first solution (i.e., lifting) and then optionally placing into the second solution or otherwise treating as described can be used. In embodiments, such a process may be performed manually. In embodiments, such a process may be performed automatically (e.g., a robot-controlled process).

[0175] In embodiments, a manufacturing platform as described above with respect to FIG. 7 can be used to alternately immerse and remove the fibrous structure adhered to the means for suspending the fibrous structure by operation of a lifting arm (e.g., lifting arm 702 of FIG. 7). In other embodiments, the user can simply move the fibrous structure adhered to the means for suspending the bioprinted fibrous structure manually from one environment to another, for example, depending on a handle (e.g., handle 218 of FIG. 2B) coupled to the means for suspending the bioprinted fibrous structure. For example, the user can position the means and the adhered fibrous structure within a solution bath and then remove the means and the adhered fibrous structure. Such an operation can be repeated any number of times.

[0176] In an embodiment, a method for bioprinting a fibrous structure includes printing at least one hydrogel fiber of continuous length around a support (e.g., support 206 in FIG. 2A) associated with means (e.g., frame 202 in FIG. 2A) for suspending two or more struts, such as 3, 4, 5, 6, 7, 8, 9, 10 or more, such as 20 or more, 30 or more, 40 or more, 50 or more, or even 100 or more bioprinted fibrous structures. In an embodiment, the means is included as part of a manufacturing platform disclosed herein and / or as part of a bioprinting system disclosed herein. In an embodiment, the method is used to generate a planar structure (i.e., one layer). In an embodiment, the method is used to generate a 3D structure (i.e., two or more layers). In an embodiment, the method further includes conformally coating the entire bioprinted fibrous structure such that its top and bottom surfaces, as well as its sidewalls, are uniformly coated. In an embodiment, the method further includes conformally coating the entire bioprinted fibrous structure a plurality of times (e.g., at least two times). Each conformally coated layer can include the same or a different coating material composition.

[0177] In an embodiment, a method for bioprinting a fibrous structure includes printing a fibrous structure comprising a core and at least one shell layer surrounding the core. In an embodiment, a coating material is applied over the entire fibrous structure to create a uniform conformal coating of the structure. The conformal coating can impart stability to the fibrous structure and / or can impart properties to the structure that optimize the interface between the fibrous structure and the host, such as anti-FBR properties, promotion of angiogenesis, etc. In an embodiment, the coating may be softer than the outermost outer shell. In an embodiment, the core, outer shell, and conformal coating can comprise from about 0.1% to about 4% alginate. In an embodiment, the fibrous structure can comprise a core of from about 0.75% to 1.5% alginate, an outer shell of from about 1.5 to 2.5% alginate, and a conformal coating of from about 0.2 to 2.5% alginate, such as from about 0.2 to 0.75% alginate.

[0178] In embodiments where two coatings are applied, it is within the scope of the present disclosure that the innermost coating may be harder than the outermost coating. In an embodiment, the core, outer shell, and conformal coating can comprise from about 0.1% to about 4% alginate. In an embodiment, the fibrous structure can comprise a core of from about 0.75% to 1.5% alginate, an outer shell of from about 1.5 to 2.5% alginate, an inner conformal coating of from 1.5% to 2.5% alginate, and an outer conformal coating of from about 0.2 to 2.5% alginate, such as from about 0.2 to 0.75% alginate. Such examples are intended to be illustrative.

[0179] In an embodiment, the bioprinted fiber structure fabricated by the method disclosed herein can be segmented / compartmentalized along at least a portion of the length of the fibers that make up the bioprinted fiber structure, preferably continuous fibers. Details of the generation of the segmented / compartmentalized bioprinted fiber structure are described in U.S. Provisional Patent Application No. 63 / 192552, the contents of which are hereby expressly incorporated by reference in their entirety. In an embodiment, the fiber consists of a core and an outer layer, and is referred to herein as a core-shell fiber, and the core and / or the outer layer are segmented / compartmentalized along at least a portion of the length of the fiber. In an embodiment, the fiber consists of a core, at least one inner shell layer, and at least one outer shell layer, and is referred to herein as an annular fiber, and any one or more of the core, the inner shell layer(s), and / or the outer shell layer(s) are segmented / compartmentalized along at least a portion of the length of the fiber. In an embodiment, one or more segments / compartments of the core and / or the shell layer may consist of a bio-derived substance (e.g., cells).

[0180] Compartment sizing may be a function of one or more variables including, but not limited to, the size of the tissue fibers (e.g., length and / or diameter), the type of fiber (e.g., core-shell fiber, annular fiber), the type of material used in the process of tissue fiber generation, etc., regardless of whether the generated fiber structure is covered one or more times. In some embodiments, the fiber may consist of at least two segments / compartments containing a bio-derived substance, and other segments adjacent to the at least two segments / compartments do not contain a bio-derived substance. For example, in the case of core-shell type fibers, at least two segments containing a bio-derived substance may be included in the core. In another example, in the case of annular fibers, at least two segments containing a bio-derived substance may be included within the first shell layer. In embodiments, the segment(s) containing a bio-derived substance may have a longer length(s) compared to the segment(s) lacking a bio-derived substance. In embodiments, the segment(s) containing a bio-derived substance may be substantially similar in terms of length compared to the segment(s) lacking a bio-derived substance. In embodiments, the segment(s) containing a bio-derived substance may be of a shorter length than the segment(s) lacking a bio-derived substance. In embodiments, the segment(s) containing a bio-derived substance for a particular tissue fiber need not be of substantially the same length, but instead, different segments may consist of different lengths. In embodiments, the segment(s) lacking a bio-derived substance for a particular tissue fiber need not be of substantially the same length, but different segments may consist of different lengths. In some embodiments, the spacing between compartments / segments containing a bio-derived substance (e.g., cells) in the tissue fibers of the present disclosure may be between 1 and 5 mm, such as 1 mm, 2 mm, 3 mm, 4 mm, or 5 mm.

[0181] Segments / compartments composed of biogenic substances may contain, for example, cells of a specific density. In embodiments, the density may be the same between compartments. In embodiments, the density may be different between compartments. In embodiments, the biogenic substances between compartments may be the same or different. In embodiments, the density of the biological material can be selected as a function of one or more of a specific application (e.g., treatment of a specific disease / illness), cell survival determinants, materials containing biogenic substances (e.g., biocompatible materials), etc. As an example, the biological agent may contain islets of the pancreas. Other biogenic substances (e.g., hepatocytes) can be used in the tissue fibers of the present disclosure at the same or different densities.

[0182] In embodiments, one or more segments / compartments containing biogenic substances may be adjacent to a segment containing, for example, a material having immunoprotective properties. For illustrative purposes and without limitation, immunoprotective hydrogel materials may include, for example, but are not limited to, functionalized alginates such as methacrylated alginate, alginate furan, alginate thiol, alginate maleimide, and covalently clickable alginates (e.g., alginates blended with DMAPS-Ald and / or DMAPS-Hzd). For example, in the case of core-shell fibers having two or more segments with a core containing biogenic substances, the two or more segments may be adjacent to other segments containing the immunoprotective materials disclosed herein. In other embodiments, two or more segments containing biogenic substances may be adjacent to other segments, for example, not containing immunoprotective materials, without departing from the scope of the present disclosure. The same logic applies to the circular fibers of the present disclosure. For example, the circular fibers may consist of two or more segments / compartments composed of biogenic substances, and each of the two or more segments / compartments may be adjacent to a segment incorporating, for example, the immunoprotective materials of the present disclosure. In other embodiments, two or more segments containing biogenic substances may be adjacent to segments, for example, not containing immunoprotective materials, without departing from the scope of the present disclosure.

[0183] Method of Use Aspects of the method include providing one or more input materials dispensed via a dispensing orifice. In some embodiments, one or more cell types are compatible with the input material and are optionally dispensed into the input material. In some embodiments, the sheath fluid functions as a lubricant to lubricate the movement of the input material (e.g., within a microfluidic printhead). In some embodiments, the sheath fluid includes a crosslinking agent to solidify at least a portion of the hydrogel before or while the hydrogel is dispensed from the dispensing orifice. In some embodiments, the crosslinking agent can be included in the input material, such as an input material corresponding to the core of the fibers of the present disclosure.

[0184] Aspects of the method include transmitting a design drawing to a 3D printer. In some embodiments, the transmission can be realized, for example, by a programmable control processor. In some embodiments, the method includes controlling the relative positioning of the dispensing orifice and the receiving surface in three-dimensional space and simultaneously dispensing, from the dispensing orifice, the input material and, in some embodiments, the sheath fluid alone or in combination. In some embodiments, the materials to be dispensed are dispensed coaxially such that the sheath fluid surrounds the input material. Such a coaxial configuration enables the crosslinking agent in the sheath fluid to solidify the input material, whereby, in turn, the solidified fiber structure is dispensed via the dispensing orifice.

[0185] In some embodiments, the method is depositing a first layer of the fiber structure dispensed via means (e.g., frame 202 of FIG. 2) for suspending the bioprinted tissue fibers, the first layer including the configuration of the fiber structure specified by the design drawing, and repeating the deposition step to deposit subsequent fiber structures onto the first layer and subsequent layers, thereby depositing layer upon layer on top of the layers of the dispensed fiber structure in the geometric arrangement specified by the design drawing to generate a 3D structure.

[0186] In some embodiments, a plurality of input materials, such as a plurality of hydrogels, at least some of which contain one or more cell types, are deposited in a controlled order, thereby enabling a controlled arrangement of the input materials and deposition of the cell types in a geometric arrangement specified by a design drawing.

[0187] In some embodiments, the method includes removing excess fluid from the receiving surface and / or from the surface of the dispensed fibrous structure. For example, the step of removing excess fluid can be performed continuously throughout the printing process, thereby removing excess fluid, which otherwise may prevent the deposition of the fibrous structure dispensed in the geometric arrangement provided by the design drawing. Alternatively, the step of removing excess fluid can be performed intermittently throughout the printing process, continuously or simultaneously with one or more deposition steps. In some embodiments, the removal of excess fluid is achieved by withdrawing the fluid from the receiving surface and / or from the surface of the dispensed fibrous structure. In some embodiments, the removal of excess fluid is achieved by withdrawing the excess fluid through a receiving surface having pores sized to allow passage of the fluid. In some embodiments, the removal of excess fluid is achieved by providing a fluid that evaporates after being dispensed from the dispensing orifice.

[0188] Aspects of the present invention include a method of fabricating a 3D structure that includes one or more input materials. The 3D structure is used to mimic the normal function of tissue within a subject that may be spaced apart or damaged.

[0189] As described above, Cd 2+ , Ba 2+ , Cu 2+ , Ca 2+ , Ni 2+ , Co 2+ , or Mn 2+ Any suitable divalent cation, including but not limited to, can be used in combination with the subject method to solidify the chemically crosslinkable input material. In a preferred embodiment, Ca 2+is used as a divalent cation. In a preferred embodiment, the chemically crosslinkable input material is contacted with a solution containing Ca 2+ to form a solidified fibrous structure. In some embodiments, the concentration of Ca 2+ in the sheath fluid ranges from about 80 mM to about 140 mM, such as about 90, 100, 110, 120, or 130 mM.

[0190] In certain embodiments, the input material solidifies in less than about 5 seconds, such as less than about 4 seconds, less than about 3 seconds, less than about 2 seconds, or less than about 1 second.

[0191] Aspects of the present invention include methods of using software tools to deposit one or more input materials in a patterned manner to form layers of solidified structures that are formed into multi-layer 3D tissue structures. In some embodiments, the multi-layer 3D tissue structure comprises a plurality of mammalian cells. Advantageously, by adjusting the components of the subject input material (e.g., mammalian cell type, cell density, matrix component, active substance), the subject method can be used to create multi-layer 3D tissue structures that have an accurately controlled composition at any particular location within the three-dimensional space. Thus, the subject method facilitates the generation of complex three-dimensional tissue structures.

[0192] All patents and patent publications referred to herein are hereby incorporated by reference in their entirety.

[0193] Examples The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention and are not intended to limit the scope that the inventors regard as their invention. Efforts have been made to ensure the accuracy of the numerical values (e.g., amounts, temperatures, etc.) used, but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0194] Example 1. Bioprinted Fibrous Structure This example demonstrates the ability to manufacture the bio-printed fiber structure of the present disclosure, which is suspended during one or more of its printing process, patterning process, and / or post-printing process. FIG. 9A shows a frame 902 (substantially similar to frame 202 in FIG. 2A) having struts 906 (substantially similar to struts 206 in FIG. 2A). Further shown is a fiber structure 908, which was generated by printing fibers around opposing struts that are continuous both vertically and horizontally to produce the illustrated lattice-type structure. FIG. 9B is an image of the fiber structure 910 after the bio-printed fiber structure 908 has been completely covered and then removed from the frame after the structure has been fully suspended.

[0195] Example 2. Immersion Printing This example demonstrates that the bio-printed fiber structure can be printed using the means for suspending the bio-printed fiber structure disclosed herein, which is immersed in a cross-linking agent solution bath during printing. FIG. 10A shows a bio-printed fiber structure printed and subsequently coated in a cross-linking solution bath using the means for suspension disclosed herein. FIG. 10B shows an enlarged view of a portion of the structure shown in FIG. 10A. FIG. 10C shows a photomicrograph of the fiber structure printed in the cross-linking solution bath, and FIG. 10D shows a photomicrograph of the fiber structure that was not printed in the cross-linking solution bath but was immersion-coated after printing. This example shows that a device in which the fiber structure is printed in a cross-linking solution bath using the suspension means disclosed herein (e.g., the frame shown in FIGS. 2A - 2D) has improved fidelity with respect to less shape deformation compared to a device in which the fiber structure is not printed in the cross-linking solution bath.

[0196] Example 3. Print Optimization and Viability Study of Small Devices This example included test conditions of a 4-layer fiber structure of 10×10 mm for a control condition of a 2-layer fiber structure of 18×18 mm. FIG. 11A is a schematic diagram of a 10×10 mm fiber structure. FIG. 11B is an image of the coated 10×10 mm fiber structure attached to the frame. FIGS. 11C to 11D show the coated 10×10 mm fiber structure detached from the frame.

[0197] The fiber structure was coated with 0.5% SLG100 (alginate). The cell dose to the structure was 3K IEQ HepG2 aggregates. Live / dead staining was evaluated at 0 days after printing (FIG. 12A) and 5 days after printing (FIG. 12B).

[0198] This example also included tests of a small device (10×10 mm, 4-layer device-coated fiber structure) having a coating of 0.5% SLG100 and a core of either 1.5% SLG100 containing HA or 1.5% SLG100. For each case, the cell dose was 3K IEQ HepG2 aggregates or primary rat islets (PRI). FIGS. 13A to 13B show images of the 10×10 mm fiber structure on the frame (FIG. 13A) and detached from the frame (FIG. 13B) after coating. The stability data are summarized in FIG. 14. FIG. 15A is an image of Structure 1 (HA-containing core), FIG. 15B is an image of Structure 2 (HA-containing core), FIG. 15C is an image of Structure 3 (HA-containing core), FIG. 15D is an image of Structure 1 (normal core), FIG. 15E is an image of Structure 2 (normal core), and FIG. 15F is an image of Structure 3 (normal core).

[0199] This example also included tests on the viability and functionality of a coated 10×10 mm fiber structure carrying PRI (coating 0.5% SLG100, cell dose: 3K IEQ PRI). Live / dead staining was evaluated at 0 days after printing (FIG. 16A) and 3 days after printing (FIG. 16B).

[0200] Example 4. Stability Test of Frame vs. Mesh Device This example demonstrates that the stability of the printed device is enhanced by using the frame of the present disclosure as compared to a device printed on a mesh instead of a frame (i.e., without using a frame).

[0201] Experimental design In this example, the coated fibrous structure tested was 18×18 mm and had a two-layer thickness. The coated fibrous structure included a core (1.5% SLG100), a shell (2% SLG100), and a conformal coating (0.5% SLG100). The core was printed at a flow rate of 115 μL / min, the shell was printed at 80 μL / min, and the sheath flow (i.e., the crosslinking agent solution) was 55 μL / min when dispensed from the print head. Three structures were printed using the frame disclosed herein, while the other three structures were printed on a mesh instead of a frame. The conformal coating was added to the bioprinted fibrous structure while attached to the frame (device dependent on the frame for printing) or added while the bioprinted fibrous structure was placed on the mesh (device printed instead of a frame).

[0202] Stability test The stability test was conducted as follows. Each coated fibrous structure was cultured in PIMS medium for 3 days in a 50 mL conical tube containing 15 mL of medium. Each coated fibrous structure was subjected to an orbital shaking test at 125 rpm for 30 minutes or vehicle transportation for 30 minutes. Next, each coated fibrous structure was poured into a Petri dish and washed 3 times with 10 mL of saline (the saline was aspirated between each wash). To mimic the transfer of the device at the surgical site, a spatula was used to lift and move each coated fibrous structure between two Petri dishes filled with saline. This was repeated 5 times. Finally, each coated fibrous structure was transferred onto a moistened plastic wrap, and a wand was used to move each device 3 times from one edge of the wrap to the other side (mimicking the repositioning of the device on the greater omentum).

[0203] Results All three coated fiber structures printed and coated by relying on the frame disclosed in this specification passed the stability test (compared with FIGS. 17 and 18D to FIGS. 18F, FIGS. 18A to 18C). All three fiber lattice structures printed on the mesh instead of the frame failed the stability test, and the microscopic images (FIGS. 19A to 19C) revealed that the fibers in the first layer leaked from the coating layer (compare FIGS. 19B - 19C with those of FIG. 19A). FIG. 20 shows an image of another fiber structure printed on a mesh (however, uncoated) indicating a decrease in stability. The structure in FIG. 20 was created by inside - out cross - linking.

[0204] Example 5. The bioprinted cell therapy platform normalizes blood glucose control in diabetic rats We have developed a microfluidic bioprinting technology that combines biocompatible materials with clinically relevant cells to produce implantable tissues for therapeutic use. Islet cell therapy has been clinically validated for type 1 diabetes (T1D), but it relies on lifelong immunosuppression and is limited by the supply of cadaveric donor islets. We are developing a bioprinted pancreatic tissue therapeutic agent that can deliver allogeneic islets or stem - cell - derived pancreatic beta cells to T1D patients without the need for immunosuppression by encapsulating these cells in materials that support physiological function and protect these cells from direct attack by host immune cells.

[0205] This example describes a bioprocess method for packaging primary islets in bioprinted tissue implants for in vitro testing and in vivo function studies. We evaluated the ability of bioprinted human islet tissue (heterologous) to restore blood glucose control in diabetic immunodeficient mice and adapted this process to bioprinted primary rat islet tissue (allogeneic) delivered to the omentum of diabetic rats. Finally, we developed a process for scaling up and down the manufacturing process of this bioprinted pancreatic tissue for delivery of implants in large animals and humans.

[0206] Materials and Methods The major procedures performed on the animals were the implantation of the framework device outlined below. The surgery was performed after treatment with STZ.

[0207] A. Prepare the animals for surgery. It followed the general concepts of "Rodent Anesthesia" (SOP ACC-01-2017), "Analgesia for Adult Mice and Rats Meloxicam SOP" (TECH19), and "Local Anesthesia / Analgesia in Adult Mice and Rats Bupivacaine SOP" (TECH16). The procedures are described below. A1. Place the animal on top of a heating pad (the temperature should be about 38°C) in the induction chamber and induce anesthesia with isoflurane. Flush the chamber and transfer the animal to the maintenance circuit on the nose cone and heat support to assist with heat and maintain isoflurane anesthesia. A2. Administer small droplets of lubricating eye gel to both eyes. Place the animal in the prone position and administer a supportive therapy solution subcutaneously at 20 mL / kg in the form of 0.9% saline or lactated Ringer's solution (LRS). Use 25G of various syringe sizes depending on the dose. A3. Administer 1 mg / kg of Metacam subcutaneously. A4. Administer 0.05 mg / kg of Buprenorphine subcutaneously. A5. While turning the animal over, place a paper towel underneath and collect all the shaved hair. Shave the abdominal skin of the animal with a clipper. A6. Remove the dry gauze, remove all the hair around the animal, and simultaneously pull out the paper towel. A7. Once the hair is cleared, prepare one gauze or cotton swab dipped in soap and clean the shaved area. The gauze should be wet but not dripping and can be wrapped around the finger. When cleaning the shaved area outward in a circular motion, bubbles should be visible. Leave the soap on the animal for about 30 seconds and then wipe it off with alcohol. Wipe the shaved area outward in a circular motion with a gauze or cotton swab dipped in 8.70% alcohol. Use a new gauze or cotton swab dipped again in soap, but this time to remove / scrub off the skin oil. After leaving it for 30 seconds, proceed to the next step. A9. Clean the area with a new gauze or cotton swab dipped in alcohol. A10. Inject a local anesthetic as a line block at the planned incision site. Lift the skin and insert the needle subcutaneously under the skin. Withdraw the needle while injecting to form a "bleb". A110. Perform an additional subcutaneous treatment once (using a gauze dipped in soap and then alcohol again). Pinch the animal's toes to ensure that the animal is properly anesthetized. Change gloves and wash hands with soap. A12. Monitor the color of the extremities (should be pink), respiratory rate, and depth throughout the procedure, and pinch the toes every 5 minutes.

[0208] B. Preparation of surgical instruments B1. Wear clean test gloves. This surgery is performed using aseptic tip techniques, and the gloved hands must remain sterile and not come into contact with any surface. B2. Open the sterile pack on the counter, aseptically remove the sterile field / wrap folded in half, and open it so that the inner side (sterile field) faces up when placing it near the operating table. B3. Prepare a pair of sterile forceps to transfer all instruments and consumables in the pack to the sterile field wrap. Only the sterile parts of the instruments can enter the sterile field. B4. Take the "Glad Press and Seal Wrap", pull out the piece that extends the first 6 inches out and place it over the animal. Avoid using the edges as they are contaminated. Do not touch the top surface with your fingers. Use a pair of forceps to pick up the cotton pad and help attach the press and seal to the animal. To pick up the press and seal, use a sterile 25G needle to puncture near the surgical incision area, grasp it with forceps, and make a hole over the planned incision site. Do not let non-sterile items contact the upper surface of the surgical drape. B6. Hold the animal with forceps and confirm that it is at the surgical level of anesthesia. C. Surgical procedure.

[0209] It was carried out in accordance with the general concept of "Rodent Survival Surgery" (SOP ACC-02-2017). All surgical procedures on immunodeficient rats were performed within a biosafety cabinet of a laminar flow clean air workstation. The procedures are described below. Use toothed forceps to pick up the skin. Use the blade of a surgical scalpel to make a 20-mm incision along the midline of the skin about 2 cm below the xiphoid process. Once the skin incision is complete, grasp and lift the muscle, first use the blade of a surgical scalpel to make a puncture opening, and then make a 20-mm incision using scissors. Use a self-retaining tissue retractor to keep the abdominal incision open. Sterilely place sterile gauze on the abdominal skin caudal to the opening to prevent the greater omentum from coming into direct contact with the skin. Identify the larger omentum and gently extend the greater omentum out of the opening using a pair of tissue forceps. Sterilely place the bioprinted implant in the center of the exposed omentum. Note: The bioprinted implant consists of a non-reactive and non-rigid polymer with no known biocompatibility issues. Each device is 20×20×2.5 mm and may contain cells, but is specifically designed to prevent cell release. One device is implanted per animal. The implant is printed using sterile medical-grade components. (Cells are incorporated during the printing process.) After printing, the device is maintained in standard cell culture conditions (medium, 37°C / 5% CO2) within 4 days prior to implantation. Immediately before implantation, the device is washed with a sterile isotonic solution such as saline or Ringer's buffer to remove all traces of the medium. C7. Fold the free end of the greater omentum over the implant and suture both sides of the mesh using a fine non-absorbable monofilament (e.g., 6-0 nylon or prolene) to create a closed pouch. C8. Gently slide the greater sac back into the abdominal cavity and close the incision with a continuous suture for the muscle layer and skin layer or a subcutaneous suture, respectively. C9. Moisten the 5-0 absorbable suture with saline. Use one suture pack per rat. Hold the needle vertically using a pair of needle drivers. Ensure that the suture does not touch the non-sterile area of the needle holder or forceps. C10. Insert the needle on one side of the muscle and withdraw it from the other side of the muscle. Follow the direction of the needle when withdrawing it. Tie a running stitch and repeat this knot a total of 3 times. Cut the end, leaving about 2-3 mm of the suture. Repeat until the muscle area is closed. C11. Perform a subcutaneous closure of the skin. Insert the needle just below the skin layer on one side of the skin and withdraw it from the opposite side of the skin (deeper but under the skin layer). Insert the needle on the opposite side of the skin and move to the surface as you tie the knot. Ensure that the needle does not come out above the skin. Tie 3 running stitches. C12. Once the skin layer is closed, take a 25G needle and immerse it in Gluture. Take the needle covered with Gluture and place it on top of the sutured skin. Pinch the skin around the needle using a pair of forceps and slowly remove the needle. This closes the sutured area properly. Recovery of rats: C13. Turn off isoflurane and continue to give oxygen to the rats via the nose cone. C14. Gently remove the surgical drape and remove the adhering blood from the rats. C15. When the right reflex of the rats has recovered, place the rats in a warmed recovery cage (cover the bare bottom of the cage with paper towels). C16. Monitor the rats in the recovery cage until they have fully recovered from anesthesia and can maintain their body temperature without additional heat (i.e., can eat, drink, walk normally, climb into the hut, and groom themselves). C17. When fully recovered, return the rats to the normal housing cage with a cage mate.

[0210] D. Analgesia plan D1. Preoperative analgesics: The rats are administered a local anesthetic (bupivacaine 0.5 mg, 200 μl of a 2.5 mg / ml solution) at the incision site before tissue dissection and receive injections of an NSAID (meloxicam 1 mg / kg, SC) and buprenorphine (0.05 mg / kg, SC). Rats with induced liver disease are given a combination of oral ibuprofen and low-dose buprenorphine to account for the reduced liver metabolism in these animals with liver disease. Oral ibuprofen (30 mg / kg, resuspend ibuprofen ligel capsules in water by vortexing, protect from light, replace every 3 days) is administered to the rats at the time of surgery according to ECH 09b Oral dosing (Gavage) in Rats. D2. Postoperative analgesics: Day 1 and Day 2: Meloxicam (1 mg / kg, SC, SID) and buprenorphine (0.02 mg / kg, SC, BID) These procedures were in accordance with TECH 16 (Local Anesthesia / Analgesia in Adult Mice and Rats Bupivacaine SOP) and TECH 19 (Analgesia for Adult Mice and Rats Meloxicam SOP). Rats with induced liver disease were given a combination of oral Ibuprofen and low-dose Buprenorphine to account for the reduced liver metabolism in these liver disease animals. Oral Ibuprofen (30 mg / kg, Ibuprofen liquid gel capsules were resuspended in water by vortexing, protected from light, and replaced every 3 days) was administered to the rats 6 hours after surgery in accordance with ECH 09b Oral dosing (Gavage) in Rats. Overnight, the animals continued to receive Ibuprofen (1 mg / mL) in their drinking water. The animals received subcutaneous injections of Buprenorphine (0.05 mg / kg) 24 and 48 hours after surgery.

[0211] E. Administration of prophylactic antibiotics The bioprinted implants were prepared aseptically using sterile materials and reagents. Also, all surgical procedures were performed aseptically. However, to reduce the potential risk of infection, a prophylactic antibiotic (100 μg / mL of Enrofloxacin in the drinking water) was added to the rats' drinking water from 3 days before surgery until 3 days after surgery. Enrofloxacin is excreted mainly by the kidneys and is also suitable for rats with liver disease.

[0212] Other procedures F. Blood sampling Blood samples were collected from rats weekly until the end point of the study. Blood was collected from the lateral saphenous vein using TECH02 SOP "Blood collection from the lateral saphenous vein in mice and rats". The maximum weekly blood sample volume was 150 ul, which is within the acceptable limits of serial blood collection defined by "UBC ANIMAL CARE COMMITTEE POLICY 006, Policy on Acceptable Methods of Rodent Blood Withdrawal".

[0213] G. Administration of Streptozotocin for Induction of Diabetes Streptozotocin (STZ) destroys pancreatic insulin-secreting cells, thereby inducing diabetes. Since special handling precautions are required for the use of STZ, all users and animal care technicians were informed of the risks due to the presence of SDS in the experimental area. Cages containing animals treated with STZ were marked as such for 1 week after STZ injection. STZ, supplied as a powder, was reconstituted to a concentration of 30 mg / mL immediately before injection in buffered acetic acid solution or citrate buffer (pH 4.5). To induce a model of type 1.1 diabetes (insulin deficiency), STZ was intraperitoneally injected into rats at a dose of 60 mg / kg in buffered acetic acid solution or citrate buffer (maximum volume approximately 0.5 mL for a standard 250 g rat). For IP injection procedures, TECH10b (Intraperitoneal Injection in the Adult Rat) was followed. G2. After injection, the blood glucose levels of the animals were monitored daily. Animals that developed persistent hyperglycemia (blood glucose level > 20 mmol / l in two consecutive readings) were used in the experiment. There is a risk of severe hypoglycemia in the first 24 to 48 hours. The initial cytotoxic destruction of beta-islet cells causes an excessive release of insulin into the bloodstream. To prevent fatal hypoglycemia, sucrose water was given during the induction period to reduce morbidity and mortality. To do so, 10% sucrose was added to the drinking water 48 hours after STZ injection.

[0214] H. Blood glucose measurement Blood glucose levels were measured by placing a drop of blood (≤10 μL) on a blood glucose meter test strip (Lifescan Canada or equivalent). H1. A small droplet of blood was collected from the tail vein using TECH13 (UBC ACCtail Poke SOP-Rat) and added to the test strip. H2. Gently apply pressure to the tip of the tail with a 2×2 gauze for about 10 seconds to stop blood flow.

[0215] I. Oral glucose tolerance test The main function of pancreatic islets is to secrete insulin in response to elevated blood glucose levels. To monitor the in vivo function of implanted pancreatic islets, the islets were stimulated with an oral glucose dose after a short fasting period described below. II. Fast the animals for 4 hours (e.g., 8:00 am to 12:00 pm). I2. Collect a blood sample (fasting, volume: 75 μL) I3. Using a 1 mL syringe and a flexible 20-gauge and 38 mm long feeding tube (according to TECH09 - Oral dosing in Mice and Rats), administer a glucose solution (3 g / kg, 150 μL) to the animals by forced oral administration. I4. After 30 minutes, collect another blood sample (after glucose administration, volume: 75 μL).

[0216] J. Immunofluorescence protocol for insulin and CD31 Substances: Insulin (C27C9) rabbit mAb (New England Biolabs), CD31 (PECAM-1) mouse mAb (New England Biolabs), anti-rabbit IgG, Alexa Fluor® 647 conjugate (New England Biolabs), anti-mouse IgG, Alexa Fluor® 488 conjugate (New England Biolabs). Procedure 1. Place the slides in an oven at 60 °C for 15 minutes to initiate the deparaffinization process. 2. Place the slides in a xylene-resistant holder. 3. Wash in xylene for 5 minutes (3X). 4. Wash in 100% EtOH for 5 minutes (2 times). 5. Wash in 95% EtOH for 5 minutes. 6. Wash in 80% EtOH for 5 minutes. 7. Wash in 70% EtOH for 5 minutes. 8. Wash on a shaker in PBS for 5 minutes. 9. Place the slides in a beaker and cover with antigen retrieval buffer (10 mM citrate buffer, pH 6.0). 10. While ensuring that the slides are always covered with buffer, heat in a microwave at high power for 5 minutes (2 times). 11. Using heat-resistant gloves, remove the beaker from the microwave and place it in the sink. 12. Cool the beaker under a thin stream of cold running tap water for 5 - 10 minutes (make sure the water does not directly hit the slides). 13. Wash the slides in ddH2O for 5 minutes. 14. Wash the slides in PBS on a shaker for 5 minutes. 15. Remove excess water on the slides and surround the sample with a hydrophobic pen (Super Pap pen). 16. Block the slides in a humidity chamber in 5% BSA-PBS containing 5% goat serum at room temperature for 1 hour. 17. Incubate with the primary antibody in a humidity chamber (diluted in 5% BSA-PBS containing 5% goat serum) at 4 °C. a. Insulin: 1 / 500 dilution b. CD31: 1 / 500 concentration Day 3 1. Wash the slides with PBS for 10 minutes (3 times). 2. Incubate with secondary antibodies for 1 hour at room temperature in a dark humidity chamber (diluted with 5% BSA-PBS or PBS). a. Anti-mouse: 1 / 1000 dilution b. Anti-rabbit: 1 / 1000 dilution 3. In subsequent steps, the slides must be protected from light. 4. Wash the slides with PBS for 10 minutes (3 times). 5. Mount the slides with Fluoroshield containing DAPI and seal the coverslip with clear nail polish. 6. Dry for 24 hours before imaging.

[0217] Treatment of islets and in vitro bioprinting Figure 21A shows a schematic diagram and bright-field image of bioprinted primary human islet tissue. Figure 21B shows live / dead staining of bioprinted primary islets (top: native islets, bottom: reaggregated islets) after 7 days in culture. Figure 21C shows data from a glucose-stimulated insulin secretion (GSIS) assay performed using primary human islets (n = 8) and primary rat islets (n = 10), mean + / - SEM.

[0218] Function of bioprinted pancreatic tissue in a streptozotocin-induced rodent diabetes model Immunodeficient (NSG) mice: IP implant Figure 22A shows the random-fed blood glucose measurements over 80 days after Streptozotocin (STZ) treatment and intraperitoneal (IP) implantation of bioprinted human islet tissue in NSG (NOD scid gamma) mice (n = 5). Day 0 represents the time of implantation. Figure 22B shows the human C-peptide levels measured in mouse plasma over 80 days using ELISA. Figure 22C shows data from an oral glucose tolerance test (OGTT) performed on day 80 to evaluate the dynamics of euglycemia after fasting and subsequent glucose load in NSG mice with bioprinted islet tissue or healthy non-STZ-treated control mice.

[0219] Immunodeficient (nude) rats: Omental implant Figure 23A shows the blood glucose measurements over 180 days after omental porch implantation of bioprinted rat islet tissue in nude rats (n = 2) treated with STZ. Figure 23B shows H&E (high and low magnification) and immunohistochemistry (IHC) for insulin (islets) or CD31 (endothelial cells) performed on sections of fixed bioprinted tissue explanted after 180 days.

[0220] Immunocompetent (Sprague-Dawley) rats: Omental implant Figure 24A shows the blood glucose measurements over 90 days after omental porch implantation of bioprinted Lewis rat islet tissue in Sprague-Dawley (SD) rats (n = 3) treated with STZ. Retrieval of explants and return to hyperglycemia were performed 30, 60, and 90 days after surgical implantation. Figure 24B shows H&E) and IHC for insulin (islets) or CD31 (endothelial cells) performed on sections of fixed bioprinted tissue explanted after 60 days.

[0221] Expansion of bioprinted pancreatic tissue for large animals Figure 25A is a schematic diagram showing that the biomanufacturing process involves tissue design and QC in the unique software of the bioprinted tissue, including microarchitecture and macroarchitecture, cell viability, and confirmation of island distribution. Figure 25B shows the bioprinted pancreatic tissue used in the rat study compared to the enlarged tissue of large animals. Figure 25C shows the survival rate of the bioprinted neonatal pig islets confirmed up to 14 days after printing. GSIS indicates that the function of the bioprinted tissue expands and contracts with the dose of human islets.

[0222] Conclusion We developed a process (see Figure 26) for manufacturing an implantable tissue containing bioprocessed pancreatic islets in a material that protects these allogeneic cells from attack by host immune cells. This example shows that the bioprinted pancreatic tissue can 1) maintain islet survival and function in vitro, 2) restore glycemic control in diabetic mouse and rat models, 3) support islet function and immune protection over 90 days in a diabetic rat model, and 4) can be scaled up for studies in large animal trials and ultimately for delivery to T1D patients.

[0223] Example 6. Double Immersion Coating of Bioprinted Fiber Structures In this example, the coated fiber structure tested was 16×16 mm and had a two-layer thickness and was printed on a device of the present disclosure that includes the containers described herein. The coated fiber structure included a core (1.5% SLG100 containing cells or dye), a shell (2% SLG100), an inner conformal coating (2% SLG100), and an outer conformal coating (2% Zwit-20 alginate). The structure was printed using the frame disclosed herein. The inner and outer conformal coatings were added to the bioprinted fiber structure while being attached to the frame (a device that depends on the frame for printing).

[0224] After printing a two - layer 16×16 mm fibrous structure, the structure was cross - linked for 3 minutes with 95% / 5% Ca / Ba in 15% polyethylene glycol (PEG) in pH - buffered dH 2 O. The cross - linking solution bath was removed by vacuum, and the fibrous structure was rinsed with TSC saline from the buffer solution channel on the print head. Next, all solutions were removed by vacuum, and the fibrous structure was lifted 3 - 5 mm from the receiving surface.

[0225] Next, 1 mL of a first conformal coating solution of 2% SLG100 was pipetted into the container to uniformly coat the entire fibrous structure, and it was confirmed that all fibers were covered with the solution from both the top and the bottom. The fibrous structure was incubated in the first conformal coating solution for 10 seconds, and then the excess solution was removed from the container by vacuum.

[0226] Next, 1 mL of a second conformal coating solution of Zwit - 20 alginate was pipetted into the container to uniformly coat the entire fibrous structure, and it was confirmed that all fibers were covered with the solution from both the top and the bottom. The fibrous structure was incubated in the second conformal coating solution for 35 seconds, and then the excess solution was removed from the container by vacuum.

[0227] The fibrous structure was then transferred to a 95% / 5% Ca / Ba solution bath, cross - linked for 3 minutes, and then the fibrous structure was rinsed with saline.

[0228] Photographs of the fibrous structure with the inner and outer conformal coatings were taken and shown in Figure 27. The diameter of the entire vertical fiber with both conformal coatings shown in Figure 27 was 1.004 mm. The inner conformal coating was between 40.8 μm (left, mid - point) and 50 μm (right, mid - point), and the outer conformal coating was between 88.1 μm (left, mid - point) and 90 μm (right, mid - point).

[0229] The above-described embodiments and examples are merely illustrative and are intended to be non-limiting. Those skilled in the art will be able to recognize or confirm many equivalents of specific compounds, materials, and procedures using only routine experimentation. All such equivalents are considered to be within the scope of the present invention and are encompassed by the appended claims.

Claims

1. A manufacturing platform for supporting a bioprinted fibrous structure during printing, patterning, and / or processing, wherein the platform comprises a frame having a plurality of supports on both sides of the frame for defining cavities and fixing and suspending at least one crosslinkable fiber within the frame to form the fibrous structure, and the at least one fiber of continuous length is printed around at least two, three, four, five, six, seven, eight, nine, ten or more of the supports during the 3D bioprinting process.

2. The manufacturing platform according to claim 1, wherein the support column is positioned inside the frame, and preferably the support column is positioned on a frame projection extending into the cavity.

3. The manufacturing platform according to claim 2, wherein the support columns are arranged around the frame at equal or uneven intervals.

4. The manufacturing platform according to claim 1, wherein the frame comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 support columns.

5. The manufacturing platform according to claim 1, wherein at least a portion of the crosslinkable fibers comprises a bio-derived material.

6. The manufacturing platform according to claim 1, wherein the frame is square, rectangular, triangular, hexagonal, octagonal, circular, or irregular in shape.

7. The manufacturing platform according to claim 1, wherein the fibrous structure includes a lattice.

8. The manufacturing platform according to claim 6, wherein the frame is coupled to a mounting bracket configured to adjust the position of the frame with respect to a receiving surface.

9. The manufacturing platform according to claim 1, wherein the frame further comprises fiber cutting grooves positioned along the cavity to allow a cutting tool to cut a portion of the fiber structure.

10. The manufacturing platform according to claim 1, further comprising a fitting groove disposed on the bottom surface of the frame and configured to receive the wall of a container or vessel on the receiving surface.

11. Means for suspending a bioprinted fibrous structure during printing, patterning, and / or processing, wherein the means for suspension comprises a frame coupled to a mounting bracket and / or a receiving surface of a bioprinting system, the frame comprising a plurality of supports surrounding the frame for fixing at least one crosslinkable fiber of continuous length that forms the fibrous structure.

12. A bioprinting system, The manufacturing platform according to any one of claims 1 to 10, or the means for suspension according to claim 11, At least one dispensing orifice for dispensing the at least one crosslinkable fiber onto the receiving surface, A positioning unit for positioning the receiving surface in three-dimensional space relative to the dispensing orifice, the positioning unit being operably coupled to either the receiving surface or the at least one dispensing orifice, Dispensing means for dispensing the at least one crosslinkable fiber from the at least one dispensing orifice, The bioprinting system comprising the above.

13. The bioprinting system according to claim 12, wherein the manufacturing platform or the means for suspension is suspended on the receiving surface.

14. The bioprinting system according to claim 12, wherein the receiving surface includes a porous material.

15. The bioprinting system according to claim 12, wherein the receiving surface comprises a container containing liquid.

16. The bioprinting system according to claim 13, further comprising a programmable control processor for controlling the positioning components and for controlling the flow rate of one or more fluids via the dispensing means.

17. The bioprinting system according to claim 13, wherein the dispensing means comprises at least one pump, and optionally the at least one pump comprises a pump assembly comprising a plurality of pumps positioned radially on a mounting bracket.

18. The bioprinting system according to claim 12, further comprising at least one print head having multiple microfluidic printing channels for selectively providing each of multiple materials.

19. The bioprinting system according to claim 12, further comprising a vacuum chuck disposed on the receiving surface and an integrated container formed by a wall protruding from the upper surface of the vacuum chuck and defining the outer circumference of the container, wherein preferably the wall is configured to be inserted into a fitting groove at the bottom of the frame.

20. A method for bioprinting fibrous structures, To provide the system according to claim 12, and to dispense the crosslinkable fibers in a continuous length around a plurality of the supports on the frame of the manufacturing platform in order to generate the fibrous structure, The method comprising the above.

21. Adding a conformal coating to the entire outer surface of the fiber structure while the fibers remain attached to the frame; Transporting the fibrous structure from one location to another while it remains attached to the frame; and / or, The method according to claim 20, further comprising housing the fiber structure while the fiber structure remains attached to the frame.

22. A bioprinted fiber structure produced by the method of claim 20, comprising a continuous length crosslinkable fiber containing at least one bio-derived material, wherein the crosslinkable fiber comprises a solid core and at least one outer shell layer surrounding the solid core, and the bioprinted fiber structure comprises at least two lattices / grids formed by the continuous crosslinkable fibers.

23. The bioprint fiber structure according to claim 22, wherein each layer has a thickness of approximately 0.050 mm to approximately 3 mm.

24. The bioprinted fiber structure according to claim 23, having a packing density between approximately 10% and approximately 90%, or between approximately 20% and approximately 80%, or between approximately 30% and approximately 70%, or between approximately 40% and approximately 60%, preferably approximately 30%, approximately 40%, approximately 50%, or approximately 60%.

25. The bioprinted fiber structure according to claim 22, having an interfiber distance between approximately 1,000 and 2,000 μm, such as approximately 1,400 to 1,600 μm or approximately 1,500 μm.

26. The bioprinted fiber structure according to claim 22, wherein the solid core comprises at least one bio-derived material, and optionally the solid core is partitioned along the length of the fiber.

27. The bioprinted fiber structure according to claim 22, comprising at least one internal shell layer surrounding the solid core, wherein the at least one internal shell layer comprises at least one bio-derived material, and optionally, the solid core and / or the at least one internal shell layer are partitioned along the length of the fiber.

28. The bioprinted fiber structure according to claim 22, further comprising at least one conformal coating.

29. The bioprinted fiber structure according to claim 28, comprising a single conformal coating.

30. The bioprinted fiber structure according to claim 29, wherein the solid core comprises 0.75 to 1.5% alginate, the at least one outer shell layer comprises 1.5 to 2.5% alginate, and the coating comprises 0.2 to 0.75% alginate.

31. The bioprinted fiber structure according to claim 28, comprising an inner conformal coating and an outer conformal coating.

32. The bioprinted fiber structure according to claim 22, wherein the at least one biological material comprises pancreatic islet cells.

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