Systems and methods for manufacturing bioprinted fiber structures

Abstract

Aspects of the present disclosure include a manufacturing platform for supporting bioprinted fibrous structures during printing, patterning and / or processing, the manufacturing platform including a frame having a plurality of columns for securing crosslinkable fibers during printing of the crosslinkable fibers, and wherein a continuous length of the crosslinkable fiber is printed around a plurality of columns 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 manner, the bioprinted fibrous structure includes a uniform outer surface and may be readily tailored and / or further processed after printing and patterning are completed.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 342,118, filed on May 15, 2022, the content of which is incorporated herein by reference in its entirety. Field of the Invention

[0003] The present disclosure generally relates to three - dimensional (3D) printing and the generation of three - dimensional biological structures from digital files. Specifically, the present invention relates to systems and methods for generating cross - linkable fibrous structures having a uniform outer surface during printing and patterning and facilitating post - printing trimming thereof. Background of the Invention

[0005] The field of tissue engineering has long sought to use a variety of materials and methods to fabricate viable synthetic structures and devices that can mimic the functions of target tissues or organs. Unfortunately, however, the practical realization of these synthetic structures still faces many significant 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). In addition, the materials used to create the synthetic structures must be biocompatible and have sufficient strength and elasticity to remain in the body for a long time without triggering an adverse immune response.

[0006] In particular, implantation in the body triggers a coordinated biological response by both the innate and adaptive immune systems against the device, the intention of which is to neutralize it, which is known as the foreign body response (FBR). The cellular response to a perceived pathogen that is too large to be phagocytosed is mediated in part by macrophages that over - express extracellular matrix (ECM) proteins such as fibronectin and also produce profibrotic factors that enhance fibroblast fibrogenesis, resulting in the formation of a fibrotic capsule around the device. Such fibrotic capsules can interfere with device function, especially when they contain therapeutic cell populations that require access to nutrient and oxygen flow to perform their intended functions.

[0007] A wide variety 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). Additionally, physical parameters of synthetic tissue structures such as shape, size, stiffness, and texture are also known to be inherent properties that cause FBR. For example, the surface of a synthetic tissue structure affects the behavior of macrophages and other immune cells because structures with no sharp edges and a smooth surface are generally more biocompatible and induce less inflammation (Mariani E et al., Int J MolSci. (2019); 20:doi:10.3390 / ijms20030636; Salthouse TN, Journal of Biomedical Materials Research Part A. (1984); 18:395 - 401). Furthermore, changes in surface roughness at the nanoscale are also associated with increased protein adsorption (Hulander M et al., Int J Nanomedicine. (2011); 6:2653 - 2666; Roach P., J Mater Sci Mater Med. (2007); 18:1263 - 1277; Scopelliti 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 nanostructure morphological features affect cell interactions (Baker DW et al., Biomacromolecules. (2011); 12:997 - 1005; Jahed Z., Biomaterials. (2014); 35:9363 - 9371).

[0008] Accordingly, there remains a need to improve both the design and the materials to accommodate the opposing purposes of immune protection and nutrient passage, and to help mitigate the FBR response. Thus, there is a need for a synthetic tissue structure that effectively balances the ability to reduce or avoid immune system recognition and / or destruction of such a synthetic tissue structure with the ability to ensure sufficient passage of oxygen and nutrients through the cells of the synthetic structure. There is also a need for a synthetic structure in which the patterning is consistent and reliable, and a method of generating the same, where the structure has sufficient strength and elasticity to remain in the body for a long time and can be easily retrieved. SUMMARY OF THE INVENTION

[0009] The present invention addresses the foregoing drawbacks in the prior art by a manufacturing platform for generating tissue fiber structures having anti-FBR properties and improved structural stability, a member for suspending a bioprinted fiber structure, a bioprinting system incorporating the member, and methods of using the same. As disclosed and demonstrated herein for the first time, 3D bioprinted fiber structures are generated in a manner that reduces or avoids contact with a receiving surface during printing, patterning, and / or processing. This in turn can significantly reduce or eliminate the introduction of surface indentations or other defects, facilitate the transportation and / or manipulation of the fiber structure, and allow conformal coating of the entire outer surface of the bioprinted fiber structure with materials that can impart desired properties such as enhanced stability and / or anti-FBR.

[0010] Aspects of the present invention include a manufacturing platform for supporting a bioprinted fiber structure during printing, patterning, and / or processing, wherein the platform includes a frame that defines a void and includes a plurality of posts on opposite sides of the frame for fixing and suspending at least one crosslinkable fiber within the frame to form the fiber structure, wherein during a 3D bioprinting process, continuous lengths of the at least one fiber are printed around at least two, three, four, five, six, seven, eight, nine, ten, or more of the posts.

[0011] In an embodiment, the posts are positioned inside the frame; preferably, wherein the posts are positioned on frame protrusions that extend into the void. In an embodiment, reliance on the protrusions minimizes contact between the fiber and the frame.

[0012] In an embodiment, the posts are spaced apart evenly or unevenly around the frame.

[0013] In an embodiment, the frame includes at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 posts, preferably having a height between about 0.5 mm and about 50 mm.

[0014] In an embodiment, at least a portion of the crosslinkable fibers comprises a biomaterial.

[0015] In an embodiment, the frame is triangular, rectangular, hexagonal, octagonal, circular, and the like.

[0016] In an embodiment, the fibrous structure comprises a grid.

[0017] In an embodiment, the frame is coupled to a mounting bracket configured to adjust the position of the frame relative to a receiving surface. In an embodiment, the frame further comprises a fiber cutting groove positioned adjacent to the void to permit a cutting tool to cut a portion of the fibrous structure. In an embodiment, the frame further comprises a mating groove disposed on the bottom surface of the frame and configured to receive a wall of a corresponding container or vessel on the receiving surface.

[0018] Aspects of the present invention include a member for suspending a bioprinted fibrous structure during printing, patterning, and / or processing, wherein the member 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 posts that surround the frame to secure at least one crosslinkable fiber of continuous length to form the fibrous structure.

[0019] Aspects of the present invention include a bioprinting system. In an embodiment, the bioprinting system comprises a manufacturing platform as disclosed herein, or a member for suspending a bioprinted fibrous structure as disclosed herein. In an embodiment, the bioprinting system comprises at least one dispensing orifice for dispensing the at least one crosslinkable fiber onto the receiving surface. In an embodiment, the bioprinting system comprises a positioning unit for positioning the receiving surface in three-dimensional space relative to the dispensing orifice, the positioning unit being operably coupled to the receiving surface or the at least one dispensing orifice. In an embodiment, the bioprinting system comprises a dispensing member for dispensing the at least one crosslinkable fiber from the at least one dispensing orifice.

[0020] In an embodiment of the bioprinting system, the manufacturing platform or the member for suspension is suspended above the receiving surface.

[0021] In an embodiment, the receiving surface comprises a porous material.

[0022] In an embodiment, the receiving surface includes a vessel containing a liquid (e.g., a crosslinker bath or an optional dip coating liquid), and the vessel is preferably positioned on or around a vacuum chuck. In an embodiment, the receiving surface includes a vacuum chuck and an integrated container formed by a wall that projects from the top surface of the vacuum chuck and defines the perimeter of the integrated container. In an embodiment, the wall of the integrated container is configured to be inserted into a mating groove in the bottom of the frame, preferably in a shape - fitting manner to prevent fluid leakage from the wall when the frame is placed on the container.

[0023] In an embodiment, the bioprinting system further includes a programmable control processor for controlling the positioning component and for controlling the flow rate of one or more fluids via the dispensing member.

[0024] In an embodiment, the dispensing member includes at least one pump; optionally, the at least one pump includes a pump assembly that includes a plurality of pumps positioned in a radial array on a mounting bracket.

[0025] In an embodiment, the bioprinting system further includes at least one print head that includes a plurality of microfluidic printing channels to selectively dispense a corresponding plurality of materials.

[0026] 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 the crosslinkable fiber around a plurality of the posts on the frame of the manufacturing platform to generate the fibrous structure.

[0027] In an embodiment, the method further includes adding a conformal coating to the entire outer surface of the fibrous structure while the fiber remains attached to the frame.

[0028] 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.

[0029] In an embodiment, the method further includes storing the fibrous structure while the fibrous structure remains attached to the frame.

[0030] Aspects of the present invention include a bioprinted fibrous structure made by the method of the present disclosure. In an embodiment, the bioprinted fibrous structure includes a continuous length of the crosslinkable fiber including at least one biomaterial, wherein the crosslinkable fiber includes a solid core and at least one outer shell layer surrounding the solid core, and wherein the bioprinted fibrous structure includes at least two layers of a grid / mesh formed by the continuous crosslinkable fiber.

[0031] In an embodiment, the thickness of each layer is from about 0.050 mm to about 3 mm.

[0032] In an embodiment, the packing density of the bioprinted fiber structure is 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%.

[0033] In an embodiment, the solid core comprises at least one biomaterial, optionally wherein the solid core is segmented along the length of the fiber.

[0034] In an embodiment, the bioprinted fiber structure comprises at least one inner shell layer surrounding the solid core, the at least one inner shell layer comprising at least one biomaterial; optionally wherein the solid core and / or the at least one inner shell layer are segmented along the length of the fiber.

[0035] In an embodiment, the bioprinted fiber structure comprises at least one conformal coating.

[0036] In an embodiment, the solid core comprises about 1.5% alginate, the at least one outer shell layer comprises about 2.0% alginate, and the coating comprises about 0.5% alginate.

[0037] In an embodiment, the bioprinted fiber structure comprises an inner conformal coating and an outer conformal coating.

[0038] In an embodiment, the biomaterial comprises islet cells.

[0039] Other features, objects, and advantages will be apparent from the following disclosure.

[0040] Incorporated by reference

[0041] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent, or patent application is specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 An exemplary illustration depicts a printhead of a microfluidics-based bioprinting system dispensing a fiber structure onto a receiving surface.

[0043] Figures 2A to 2E An example of a member for suspending a bioprinted fiber structure according to an embodiment of the present disclosure is depicted.

[0044] Figure 3It is an illustration showing how to bioprint a fibrous structure using a member for suspending a bioprinted fibrous structure as depicted in FIG. 2.

[0045] Figure 4 It is an image of an exemplary fibrous structure composed of two layers printed via the member for suspending a bioprinted fibrous structure of FIG. 2.

[0046] Figures 5A to 5D It depicts an illustrative embodiment of the manufacturing platform of the present disclosure.

[0047] Figures 5E to 5F It shows, respectively, a manufacturing platform including an integrated container and a corresponding frame including a groove for receiving a wall of the integrated container.

[0048] Figure 5G It shows an ordered liquid coating on the bottom and top surfaces of a fibrous structure on a frame positioned on the integrated container of the present disclosure.

[0049] Figure 5H It shows a freestanding vacuum chuck and an integrated container of the present disclosure.

[0050] Figures 5I to 5K It depicts an additional illustrative embodiment of the manufacturing platform of the present disclosure.

[0051] Figure 6 It is an illustration showing how a member for suspending a bioprinted fibrous structure, such as Figures 2A to 2C , can be used to print fibers into a bath solution.

[0052] Figure 7 It is an illustration of a manufacturing platform including a vessel for bioprinting a fibrous structure into a bath.

[0053] Figure 8 It is an illustrative process flow for coating a fibrous structure produced via a member for suspending a bioprinted fibrous structure according to an embodiment.

[0054] Figure 9A It is an image of a bioprinted fibrous structure coupled to a member for suspending a bioprinted fibrous structure according to an embodiment.

[0055] Figure 9B It is the Figure 7 bioprinted fiber removed from a member for suspending a bioprinted fibrous structure.

[0056] Figures 10A to 10D It is an image of a bioprinted fibrous structure according to an embodiment of the present disclosure.

[0057] Figure 11A It is a schematic diagram of a 10x10 mm fibrous structure of the present disclosure.

[0058] Figure 11B An image of a coated 10x10 mm fibrous structure attached to a frame of the present disclosure.

[0059] Figures 11C to 11D Depicts a Figures 11A to 11B coated 10x10 mm fibrous structure separated from the frame.

[0060] Figure 12A Depicts the live / dead staining of a coated 10x10 mm fibrous structure compared to a coated 18x18 mm fibrous structure, each fibrous structure containing HepG2 aggregates, evaluated at 0 days post-printing.

[0061] Figure 12B Depicts the live / dead staining of a coated 10x10 mm fibrous structure compared to a coated 18x18 mm fibrous structure, each fibrous structure containing HepG2 aggregates, evaluated at 5 days post-printing.

[0062] Figures 13A to 13B Shows the 10x10 mm grid structure after coating on the frame ( Figure 13A ) and separated from the frame ( Figure 13B ).

[0063] Figure 14 Summarizes the stability data of coated 10x10 mm fibrous structures containing HepG2 aggregates or primary rat islets (PRI).

[0064] Figures 15A to 15C Depicts an image of three coated 10x10 mm fibrous structures with HA-containing cores.

[0065] Figures 15D to 15F Depicts an image of three coated 10x10 mm fibrous structures without HA-containing cores (i.e., SLG100 without HA).

[0066] Figure 16A Shows the live / dead staining of a coated 10x10 mm fibrous structure loaded with PRI, evaluated at 0 days post-printing.

[0067] Figure 16B Shows Figure 16A the live / dead staining of a coated 10x10 mm fibrous structure, evaluated at 3 days post-printing.

[0068] Figure 17 Is a table showing the stability data of a fibrous structure printed on a frame of the present disclosure and then coated compared to a fibrous structure replacing such a frame.

[0069] Figures 18A to 18C is an image of a fiber structure that is printed on the framework of the present disclosure and then coated, after it has undergone a stability test, the data of which is summarized at Figure 17 .

[0070] Figures 18D to 18F is an image of a fiber structure that is printed instead of the framework of the present disclosure and then coated, after it has undergone a stability test, the data of which is summarized at Figure 17 .

[0071] Figure 19A is a microscopic image of a coated fiber structure printed through the framework of the present disclosure.

[0072] Figures 19B to 19C is a microscopic image of a coated fiber structure printed instead of the framework of the present disclosure.

[0073] Figure 20 is an image depicting an in - out cross - linked, uncoated bioprinted fiber structure printed instead of the framework of the present disclosure.

[0074] Figure 21A is a schematic diagram and bright - field image of bioprinted primary human islet tissue.

[0075] Figure 21B Shows the live / dead staining of bioprinted primary islets.

[0076] Figure 21C Depicts data from glucose - stimulated insulin secretion (GSIS) assays performed using primary human and rat islets.

[0077] Figure 22A Shows the results of random - fed blood glucose measurements within 80 days after streptozotocin (STZ) treatment and intraperitoneal (IP) implantation of bioprinted human islet tissue in NSG (NOD scidγ) mice.

[0078] Figure 22B Shows the human C - peptide levels measured in mouse plasma using ELISA within 80 days.

[0079] Figure 22C Shows data from an oral glucose tolerance test (OGTT) that was performed on NSG mice with bioprinted islet tissue or healthy, untreated STZ - control mice on day 80 to evaluate the kinetics of blood glucose normalization after a fasting period and a subsequent blood glucose challenge.

[0080] Figure 23AShows blood glucose measurement results within 180 days after omental bursa implantation of bioprinted rat islet tissue in STZ-treated nude rats (n = 2).

[0081] Figure 23B Shows H&E (high and low magnification) and insulin (islets) or CD31 (endothelial cells) immunohistochemistry (IHC) performed on sections of fixed bioprinted tissue explanted at 180 days.

[0082] Figure 24A Shows blood glucose measurement results within 90 days after omental bursa implantation of bioprinted Lewis rat islet tissue in STZ-treated Sprague-Dawley (SD) rats.

[0083] Figure 24B Shows H&E and insulin (islets) or CD31 (endothelial cells) IHC performed on sections of fixed bioprinted tissue explanted at 60 days.

[0084] Figure 25A Is a schematic diagram of the biomanufacturing process of the present disclosure.

[0085] Figure 25B Shows a comparison of bioprinted pancreatic tissue for studies in rats with scaled-up tissue for large animals.

[0086] Figure 25C Shows the viability of bioprinted neonatal porcine islets confirmed up to 14 days after printing.

[0087] Figure 26 Depicts a process flow for manufacturing an implantable grid structure containing bioprosthetic islets in a material that protects allogeneic cells from host immune cell attack.

[0088] Figure 27 Depicts a fibrous structure having both an inner conformal coating and an outer conformal coating. Detailed Description

[0089] The hanging member 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-print processing. As demonstrated herein, the member and / or the fabrication platform including the member readily facilitate post-print trimming as well as storage and / or transportation of the bioprinted fiber structure. In embodiments, the present invention facilitates post-print trimming, such as, for example, a coating that can enhance stability and / or confer anti-FBR properties. In embodiments, the present invention enables the production of bioprinted fiber structures with a more uniform conformal coating, including a coating that is free or substantially free of defects that could otherwise cause FBR when implanted into a subject. In embodiments, the fiber structures generated in accordance with the teachings of the present disclosure advantageously exhibit a reduction in FBR when implanted into a subject.

[0090] Definitions

[0091] For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural and vice versa. If any definition set forth conflicts with any document incorporated herein by reference, the definition set forth below shall control. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0092] As used herein, the term "hydrogel" refers to a composition comprising a network or lattice of water and hydrophilic polymer chains.

[0093] As used herein, the term "sheath fluid" or "sheath solution" refers to a fluid that is at least partially used to encapsulate or "wrap" a material as the material 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 contains a chemical crosslinking agent. Non-limiting examples of crosslinking agents include divalent cations (e.g., Ca 2+ , Ba 2+ , Sr 2+ , etc.), thrombin, and chemicals that alter pH, such as sodium bicarbonate.

[0094] As used herein, the term "segmented / compartmentalized" refers to the discontinuous nature of a certain type of material and / or biomaterial included in the core or shell layer of the fibers disclosed herein, such as where there are intentional gaps in the deposition of the biomaterial along the fiber length. The spacing (e.g., length) between such segments / compartments can be regular (e.g., the spacing between regions of biomaterial is approximately the same), or the spacing can vary.

[0095] As used herein, the term "solid core" refers to the core of a fiber of the present disclosure, which is composed of a specific material (e.g., a hydrogel that can be crosslinked by a chemical crosslinking agent) such that the core does not include a lumen along the entire length of the fiber. The term is not intended to refer to a core that is completely impermeable along its length, as the solid core of the present disclosure may allow specific fluids, molecules, and / or ionic species to pass through the entire core.

[0096] As used herein, the term "annular fiber" refers to a fiber composed of a solid core and one or more shell layers surrounding the solid core.

[0097] As used herein, the term "biocompatible material" refers to a material into which and / or with which a biomaterial (including but not limited to cells) can be incorporated, and in which the ability of the biocompatible material to perform one or more functions on the biomaterial (e.g., cell functions, including but not limited to the secretion of bio-related molecular species, agonist / receptor binding, signal transduction, etc.) is not adversely affected.

[0098] As used herein, the term "immunoprotection" broadly refers to a design aspect of the fibers of the present disclosure that is used to reduce, prevent, or eliminate a host immune response, including, for example, immune cell invasion of the fibers after implantation of the fibers into the body (e.g., a mammalian body).

[0099] 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 can include bio-related agents, therapeutic agents, diagnostic agents, pharmaceutical agents, chelating agents, etc. A therapeutic agent or pharmaceutical agent is a therapeutic agent or pharmaceutical agent that, when administered to a subject in a manner consistent with the present disclosure, alone or in combination with additional compounds, induces a desired response (such as inducing a therapeutic or prophylactic effect). A bio-related agent is an agent that supports another biological process, such as an agent that supports cell viability.

[0100] Introduction

[0101] 3D bioprinting is an additive manufacturing process in which synthetic fibers (optionally carrying cells) are laid down in a layer-by-layer manner to obtain a multi-layered 3D structure. A variety of types of 3D bioprinting technologies have been developed, including extrusion (Panwar A et al., Molecules. (2016); 21:685; Sakai S et al., Biofabrication. (2018); 10:045007; Han HW and Hsu SH, Neural 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., LabChip. (2016); 16:3351), laser-assisted (Sorkio A et al., Biomaterials. (2018); 171:57; Pagès E et al., J.Nanotechnol.Eng.Med. (2015); 6:021006; Catros S et al., In Vivo and In SituBiofabrication by Laser-Assisted Bioprinting, 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. Among these, extrusion is one of the most common methods, in which bioink is dispensed through one or more syringes to form a layer-by-layer scaffold from the fibers.

[0102] The progress made has also contributed to the use of microfluidics-based 3D bioprinting systems (Beyer ST et al., 2013 Transducers Eurosensors XXVII 17 th Int.Conf.Solid-State Sensors,Actuators,Microsystems.IEEE,Piscataway,NJ(2013); pp. 1206 to 1209; Beyer ST et al., The 17 thInt. Conf. on Miniaturized Systems for Chemistry and Life Sciences. (2013); pp. 176-178). With these systems and technologies, multiple materials (e.g., bioinks, crosslinkers, etc.) can flow through microchannels, which can allow precise control of one or more of flow, switching, mixing, etc. When used with a sheath flow surrounding at least one internal material, microfluidic bioprinting can reduce shear stress during the printing process. Microfluidics-based 3D bioprinting can also advantageously allow material streams to intersect as they leave separate flow paths and enter a single flow path (e.g., a dispensing channel) to facilitate the generation of structures having a core surrounded by one or more shells.

[0103] Many of these bioprinting strategies involve directly printing fibers onto a receiving surface, which can be a confounding factor in post-print processing steps. For example, coatings cannot be applied to the bottom surface of a bioprinted fiber structure that remains in contact with the receiving surface, and manipulation of the structure may cause it to fall apart or otherwise compromise its integrity. Additionally, printing a fiber structure onto a surface may introduce unwanted defects or irregularities due to contact with the surface itself.

[0104] To illustrate this, Figure 1 An exemplary microfluidic printhead 100 that can be used to print a fiber structure 120 is depicted. Printhead 100 includes a plurality of reservoirs (104, 106, 108, 110) and corresponding valves (simply depicted as "102"). The valves control the flow of material to respective microfluidic channels, which each converge into a single dispensing channel 122. In this exemplary illustration, microfluidic channel 112 directs a sheath fluid containing a crosslinker solution toward dispensing channel 122; channel 114 directs a buffer solution toward dispensing channel 122; and channel 116, which receives flow from one or both of reservoirs 108 and 110, directs a hydrogel material toward 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.

[0105] A receiving surface 124 is also shown, which includes a plurality of pores 125 in the example. In an exemplary method, the sheath fluid surrounds the hydrogel solution in dispensing channel 122 such that crosslinking of the fiber structure occurs while in the dispensing channel. Any excess sheath fluid flows through receiving surface 124, as depicted by arrow 126, while fiber structure 120 is deposited on top of the receiving surface.

[0106] Figure 1 Depicts the deposition of the first layer of the fiber structure 120 by the print head 100. Additional layers can be added on top of the first layer, for example to form a grid / mesh-like structure. As can be seen, the bottom surface of the fiber structure is in contact with the receiving surface, and thus, any procedure for coating the fiber structure with the desired material cannot reach the bottom surface of the fiber structure. In some cases, attempting to move and / or manipulate the fiber structure to coat the bottom surface may result in a reduction in the fidelity of the fiber structure due to a lack of effective fiber-to-fiber adhesion. This inability to uniformly coat the entire outer surface of the fiber structure (i.e., the inability to add a conformal coating) renders it unsuitable for implantation.

[0107] In some cases, the porous nature of the receiving surface may result in indentations along the length of the resulting fibers. As additional layers are added, the indentations / irregularities become more pronounced due to the increased overall weight of the bioprinted structure. Even for non-porous surfaces, the weight of the additional layers can compromise the structural integrity of the bioprinted fiber structure. Additionally, 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 can exert a negative force on the fibers near the pores 125, which can further exacerbate the formation of indentations / irregularities. Such indentations / irregularities can be contributing factors to the occurrence of FBR after implantation.

[0108] Components for suspending a bioprinted structure

[0109] Components for suspending a fiber structure during printing and / or transportation are described herein. In this way, the fiber structure can be bioprinted in such a manner that the resulting structure is at least partially suspended during one or more of printing, patterning, and / or post-printing processing. In some examples, printing onto the receiving surface can be completely avoided. Turning to Figure 2A , depicts an exemplary component for suspending a fiber structure during printing. In this exemplary embodiment, the component for suspension includes a frame 202 having a plurality of opposing posts 206. In an embodiment, the frame and the posts 206 are composed of a single manufacturing material, but within the scope of the present disclosure, the posts 206 can be made of a material different from that of the frame 202. In an embodiment, the frame 202 and / or the posts 206 are composed of, for example, stainless steel or a dental-grade polymer (e.g., polyethylene, polymethyl methacrylate, polycarbonate, polyethylene glycol, polyurethane, hexamethyldisilazane, etc.). In an embodiment, the choice of the material composition of the posts involves minimizing or optimizing the adhesion level between the fiber structure and the posts.

[0110] As Figure 2AAs depicted, the empty frame 202 defines a void and includes a plurality of opposing posts 206 that are on opposite sides of the frame and extend generally vertically upward relative to the frame 202. In the exemplary embodiment shown, the posts 206 are positioned inside the frame 202 and more specifically on projections of the frame 202 that extend above the void in order to minimize contact between the fibers and the frame 202 during the printing process. In Figure 2A the embodiment shown, the frame 202 has a generally square shape, but other frame shapes are readily envisioned as being within the scope of the present disclosure, including but not limited to rectangular, circular, triangular, hexagonal, and octagonal. In an embodiment, the posts may be evenly spaced and on all sides of the frame, but the spacing may also be irregular, and / or opposing posts may be positioned on only two sides of the frame, depending on the application. At a minimum, a plurality of posts 206 need to be positioned on opposite sides of the frame and spaced far enough apart such that a needle or dispensing nozzle can move around / between the posts when printing a fibrous structure.

[0111] The number of posts included in a frame for suspending a fibrous structure during printing can vary and can depend on a number of variables, including but not limited to the dimensions of the corresponding frame (e.g., perimeter), the size of the dispensing needle / nozzle, the desired application, the post thickness, the post 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 greater than 50 posts, such as 100 posts or more. The post thickness (e.g., diameter in the case of a cylindrical or semi-cylindrical post) and the post height are additional variables that can be selected according to the desired application. For example, the post height can vary between about 0.1 mm and about 10 cm, preferably between about 0.2 mm and about 20 mm, such as between about 1 mm and about 4 mm.

[0112] Turning to Figure 2B , another exemplary embodiment of a frame 210 is depicted. The frame 210 includes posts 212 and corner posts 214. The frame 210 has a greater number of posts (11 posts on each side) along sides 220 and 221 and a lesser number of posts (9 posts on each side) along sides 222 and 223. In these respects, the frame 210 is substantially similar to the frame 202 (reference Figure 2A ). In other examples, each side may have the same number of posts. Additionally, although depicted as having two posts in two opposing corners, other embodiments encompass having posts in all four corners, only three corners, only one corner, or zero corners.

[0113] In an embodiment, the posts are positioned on frame projections that extend inward from the frame into the interior of the frame. With particular reference toFigure 2B , each post (212 and 214) is positioned on a protrusion 216 extending inwardly from the frame 210. In Figure 2B , each protrusion 216 is triangular, but the protrusions 216 supporting the posts (e.g., 212 and 214) can also be other shapes (e.g., rectangular, square, semi-circular). In some embodiments, the posts (e.g., 210, 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, which can be used for grasping (e.g., manually or robotically) and manipulating / moving the frame 210.

[0114] As Figure 2B shown, the frame 210 has an approximate bowl shape, where the handle 218 is attached to the upper edge 230, and the protrusions 216 extend from the lower edge 232. In an embodiment, the overall bowl shape can enable the frame 210 to be conveniently placed in a corresponding mounting structure, as will be elaborated in more detail below.

[0115] For reference, Figure 2C another frame 250 is depicted. The upper edge 252 and the lower edge 254 are clearly depicted. The posts 256 each extend from a protrusion 258 that extends inwardly from the lower edge 254. The handle 260 extends from the upper edge 252. In Figure 2C the example shown, each side has the same number of posts, where the corner posts 262 occupy two corners of the frame 250.

[0116] Depending on the frame, the bioprinted fiber structure of the present disclosure can have different packing densities. As discussed herein, the packing density is expressed as a percentage. For example, a grid structure with a fully filled fiber structure (i.e., no space) will correspond to a packing density of 100%, while a grid structure with 90% of the space not occupied by any fiber structure will correspond to a packing density of 10%. Related to the packing density is the fiber-to-fiber distance. As discussed herein, the fiber-to-fiber distance refers to the distance of the empty space between two adjacent fibers in a layer of the grid, where the fibers in the same layer are parallel to each other (see, for example Figure 4 the exemplary fiber structure depicted).

[0117] Figures 2A to 2C The frames depicted are exemplary, and other frame designs are also within the scope of the present disclosure. Figure 2D Another frame 275 is depicted, where the posts 276 are irregularly spaced. The frame 275 includes a common outer shell 277 and a void space 278. The frame 275 is shown from a top-down perspective.

[0118] In some embodiments, the frame may include fiber cutting slots positioned such that a cutting tool, such as a scalpel, can be used to cut a portion of the fiber structure, such as long initial fibers that may be "waste." The fiber cutting slots may permit alignment of the cuttings without removing the fiber structure prior to cutting the "waste" portion of the fibers. Figure 2E An illustrative frame 280 with fiber cutting slots 282 is shown in

[0119] Thus, as regarding Figures 2A to 2E shown, the frame according to the present disclosure may have a particular shape (e.g., square, rectangular, triangular, hexagonal, octagonal, circular, irregular shape, etc.). In some examples, posts (e.g., Figure 2A post 206 at Figures 2A to 2C are illustratively depicted as being evenly spaced around the frame. However, in other examples, posts (e.g., Figure 2D post 276 at

[0120] Turning to Figure 3 , a print head 302, posts 306, a frame 308, protrusions 310, dispensing channels 314, and a bioprinted fiber structure 316 are depicted. As shown, the fiber structure may be printed around the posts 306 such that the fiber structure 316 can hang within the frame during printing, patterning, and / or post-print processing. Only two opposing posts (left and right) are shown at Figure 3 for illustrative purposes. In the illustrative example depicted in Figure 3 , the fiber structure 316 is a mesh / raster structure composed of a first layer 320, a second layer 322, and a third layer (being formed) 324. Layer 322 is depicted in cross-section and is formed by printing around opposing posts of the frame 308 in an orderly manner. These posts (e.g., Figure 3 306 at Figures 2A to 2CIn some embodiments of the frame depicted to print a grid / mesh, the distance of the void space between adjacent fibers (referred to herein as the fiber-to-fiber distance) can be between about 1000 μm and 2000 μm, such as between about 1400 μm and about 1600 μm.

[0121] Go to Figure 4 , depicted are frame 202 and posts 206, as well as illustrative fiber structure 410. Fiber structure 410 is shown as being wound around each of the opposing posts 206 in a manner to produce a bioprinted grid structure that hangs within the frame. To generate the fiber structure, a first layer can be produced by moving a fiber back and forth in the direction of arrow 414 around successive opposing posts 206, and then a second layer can be produced by again moving the fiber back and forth in the direction of arrow 418 around successive opposing posts 206, and vice versa. In principle, any number of layers can be added in this manner. In an embodiment, the height of posts 206 can vary depending on the number of layers desired for a particular structure, where the higher the post height, the greater the potential number of layers. As discussed herein, the layer height is a function of the fiber diameter, and thus for the same total height of a bioprinted fiber structure, a structure composed of smaller diameter fibers will have more layers than a structure composed of larger diameter fibers. As a representative example, a 10 mm high structure composed of 0.050 mm diameter fibers will have 200 layers.

[0122] Preferably, the fiber structure is produced by winding a continuous length of fiber around at least two, three, four, five, six, seven, eight, nine, ten or more (e.g., 20 or more, 30 or more, 40 or more, 50 or more, or even 100 or more) posts during fiber structure printing.

[0123] As Figure 4 depicted, producing fiber structure 410 in the manner discussed forms loops 420. In an embodiment, the loops can be removed from the fiber structure after printing, or they can be retained as part of the fiber structure. In embodiments where the loops are excised, the loops can be excised while the fiber structure remains otherwise attached to the frame, or the fiber structure can be removed from the frame and then the loops can be excised.

[0124] In an embodiment, depending on as Figures 2A to 4Generating a bioprinted fiber structure using the depicted framework enables manipulation of the entire fiber structure without disturbing the structural integrity of the fiber structure before and / or during fiber-to-fiber adhesion within a layer. Thus, a bioprinted fiber structure coupled to the framework can be raised above a surface (e.g., a receiving surface) and coated integrally. In an embodiment, removal of the fiber structure (e.g., a fiber structure substantially similar to fiber structure 410) can be achieved simply by inverting the framework such that gravity acts on the fiber structure to release it from the framework. In some additional or alternative embodiments, a force can be applied to the underside of the framework while the framework is inverted or at least partially inverted to assist in releasing the fiber structure from the framework.

[0125] Manufacturing platform

[0126] A manufacturing platform for generating a bioprinted fiber structure as disclosed herein includes at least the aforementioned means for suspending a bioprinted fiber structure (e.g., Figure 2A framework 202 at, Figure 2B framework 210 at, Figure 2C framework 250 at, Figure 2D framework 275 at, Figure 2E framework 280 at), the means including a plurality of posts for securing at least one bioprinted fiber to form the bioprinted fiber structure. For example, in an embodiment, the means for suspending a 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.

[0127] Turning to Figure 5A , manufacturing platform 500 is depicted. Manufacturing platform 500 includes a lift arm 502 that is coupled to a mounting bracket 506. Mounting bracket 506 is configured to receive a framework 508 (e.g., similar or identical to the Figures 2A to 2D depicted framework). The lift arm 502 and, correspondingly, the mounting bracket 506 and framework 508 can be adjusted manually or robotically (i.e., in an automated manner) to adjust the height at which the framework 508 is positioned relative to a surface 510. In an embodiment, the lift arm 502 can be adjusted between a finite number of positions (e.g., 2, 3, 4, 5, 6, 8, 10). In an embodiment, the lift arm 502 can be adjusted between any number of positions. In either case, the lift arm 502 can be fixed in place once it is set in a desired position to prevent further movement of the lift arm until further adjustment is desired.

[0128] In an embodiment, the manufacturing platform includes a vacuum chuck 512. The vacuum chuck 512 includes a chuck orifice 516 that can be used in combination with a vacuum source (e.g., a vacuum pump) to evacuate the interior of the vacuum chuck 512. The vacuum chuck 512 can be placed on a surface 510 and aligned with a frame 508 and a mounting bracket 506. In an embodiment, the area of the top of the vacuum chuck 512 is substantially equal to or greater than the area corresponding to the interior of the frame 508. In an embodiment, the top of the vacuum chuck 512 is porous such that the vacuum applied through the chuck orifice 516 sucks air through the top of the vacuum chuck 512 and into the interior space of the vacuum chuck 512. In an embodiment, the top of the vacuum chuck 512 is uniformly porous throughout the top.

[0129] In an embodiment, a mesh 514 can be placed on the top of the vacuum chuck 512. The mesh 514 can be reusable or disposable. Preferably, the mesh 514 is composed of a non-adhesive, non-reactive synthetic material that can act as a passive support for a bioprinted fiber structure in some examples. Examples include, but are not limited to, nylon, polyethylene, polyethylene terephthalate, steel, glass, PEEK (polyetheretherketone), PTFE (polytetrafluoroethylene), cellulose, etc.

[0130] In an embodiment, via a lift arm 502, the frame 508 can be lifted from the surface 510 so that, for example, the vacuum chuck 512 (and optionally the mesh 514) can be placed in a desired position relative to the frame 508. Then, via the lift arm 502, the frame 508 can be lowered in the direction of the surface 510 to position the frame 508 in a desired position relative to the vacuum chuck 512 (and optionally the mesh 514), as Figure 5B illustratively depicted. It is understood that the vacuum drawn through the vacuum chuck 512 can be used to remove excess fluid (e.g., excess sheath fluid and / or excess buffer solution) that might otherwise accumulate on the mesh 514, the top of the vacuum chuck 512, and / or the surface of the bioprinted fiber structure. Thus, in Figures 5A to 5B the depicted embodiment, the vacuum chuck 512 includes a fluid removal component.

[0131] The mounting bracket (e.g., mounting bracket 506) and optionally the lift arm (e.g., lift arm 502) can be designed to accommodate a frame having a specific size. In some embodiments, the mounting bracket and the lift arm are a single unit, while in other embodiments, the mounting bracket can be detachably coupled to the lift arm.

[0132] Thus, for reference, Figure 5CDepicts another exemplary embodiment of the manufacturing platform 522 with the lifting arm 524 in the first upper position, while Figure 5D depicts the manufacturing platform 522 with the lifting arm 524 in the second lower position. As Figures 5C to 5D shown, the mounting bracket 526 is designed to accommodate the frame 528. The manufacturing platform 522 may also include vacuum suction cups and a mesh (not numbered) as discussed above with respect to Figures 5A to 5B . As is visually apparent, the manufacturing platform 522 accommodates a smaller frame than the manufacturing platform 500. In some embodiments, the mounting bracket may be detachably coupled to the lifting arm of a particular manufacturing platform to accommodate different frame configurations and / or sizes.

[0133] In an embodiment, one or more home columns 520 are included as part of the mounting bracket 526, as Figure 5D shown. The home columns 520 can be used to align, for example, the print head of a bioprinting system as disclosed herein. The home columns 520 serve as reference coordinates that can, for example, assist the bioprinter system in finding and setting the "HOME" position for printing a 3D fibrous structure. For example, the starting position can correspond to known x, y, z coordinates relative to the home columns 520. In this way, the bioprinting system can record the dimensions of a particular frame and coordinate the movement of the print head based on the reference coordinates.

[0134] In an embodiment, during a 3D bioprinting process, by using such a manufacturing platform as disclosed, at least two, three, four, five, six, seven, eight, nine, ten or more (e.g., 20 or more, 30 or more, 40 or more, 50 or more, or even 100 or more) of the columns around a frame (e.g., frame 508) are printed with at least one continuous length of a bioprinted fiber. Such manufacturing platforms can form part of a bioprinting system as disclosed herein.

[0135] In an embodiment, the top of the mesh (e.g., the mesh 514 at Figures 5A to 5B ) and / or the vacuum suction cups (e.g., the vacuum suction cup 512) may include a receiving surface for a bioprinted fiber structure printed through the columns of a frame (e.g., frame 508). In an embodiment, the receiving surface may include, for example, a surface (e.g., surface 510) that does not include a vacuum suction cup (e.g., the vacuum suction cup 512), but may optionally include some other additional or alternative fluid removal components.

[0136] Thus, in some embodiments, 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 fibrous structure is printed onto the receiving surface (e.g., mesh 514) via a frame (e.g., frame 508), and then the frame and the corresponding fibrous structure are suspended above the receiving surface for further processing, such as applying one or more coatings to the fibrous structure.

[0137] In some embodiments, the fabrication platform may include a vessel (see, e.g., Figure 7 ). In an embodiment, the vessel includes a container in which at least a portion of the fabrication platform may be placed, such as a member (e.g., Figure 5A frame 508 at Figure 5A ) for suspending at least a bioprinted fibrous structure, and the member is optionally coupled to a mounting bracket (e.g., Figure 5A mounting bracket 506 at Figure 5A ). For example, the container may include a bath used during one or more of printing, patterning, and / or post-print trimming of the bioprinted fibers. In an embodiment, the vessel is sized such that a liquid solution held by the vessel can completely immerse at least the member (e.g., Figure 5A frame 508 at Figure 5A ) for suspending the bioprinted fibrous structure, and the member is optionally coupled to a mounting bracket (e.g., Figure 5A mounting bracket 506 at Figure 5A ). In an embodiment, such a vessel can be used for immersion bioprinting, which is discussed below.

[0138] In an embodiment, as Figure 5E shown, an integrated container 530 can be formed directly on the top surface of a vacuum chuck 532 by a wall 534 that defines the perimeter of the container. The area enclosed by the wall forms the integrated container, which can hold a liquid, such as a crosslinking agent solution, a washing solution, a storage solution, a dip coating solution, etc., onto which a frame 536 can be placed. In an embodiment, as Figure 5F shown, the wall is configured to dock with a mating groove 538 on the underside of the frame 536. Preferably, the depth of the groove is such that the volume of liquid within the container can be positioned slightly above the column height of the frame. By having such a volume of liquid enclosed by the wall, a user can precisely dispense a specific volume of liquid onto the frame.

[0139] This configuration can enable the fibrous structure to be suspended within the liquid in the container without the fibrous structure contacting the vacuum chuck and / or the mesh. In an exemplary embodiment, after the frame is placed on the integrated container, the volume of liquid enclosed by the wall may be sufficient to coat at least the underside of the fibrous structure, and an additional volume of liquid may optionally be applied on top, as Figure 5GAs shown. In an embodiment, the vacuum chuck is operatively connected to a valve (540) that can be operated to allow liquid to flow out of the vessel under vacuum or to prevent liquid flow to maintain the liquid level in the vessel.

[0140] In an embodiment, the frame or a portion thereof may be coated with a hydrophobic coating (such as Parylene-C) to prevent leakage. In an embodiment, as Figure 5H shown, a vacuum chuck (542) with an integrated container can be elevated on supports and used as a separate dip-coating station for the frame (544).

[0141] Figures 5A to 5D The depicted manufacturing platform is illustrative, and other variations are also within the scope of the present disclosure. Turning to Figure 5I , another example of a manufacturing platform 570 that can form part of a bioprinting system as disclosed herein is depicted. The manufacturing platform 570 includes a frame 571, a vacuum chuck 572, and a motionless dock 574 that also includes a frame base 575. Attached to the frame 571 is a mounting bracket 577. The vacuum chuck 572 includes a peg 576. A stage 573 is also shown. The bioprinting system can move the stage 573 along the x, y, and z planes (see Cartesian coordinate system 579). The vacuum chuck 572 can rest or otherwise be fixed to the stage 573. The frame base 575 can be removably attached to the frame 571 such that when the frame 571 is not attached to the vacuum chuck 572 via the mounting bracket 577, the frame base holds the frame 571 in place. In this example illustration, the peg 576 attached to the vacuum chuck 572 is positioned in the topmost slot in a groove 578 such that the frame 571 is directly positioned on top of the vacuum chuck 572.

[0142] Turning to Figure 5J , the manufacturing platform 570 is shown where the frame 571 is attached to the frame base 575 and the stage 573 has been moved away from the frame 571. Figure 5K The manufacturing platform 570 is depicted where the stage 573 has been moved in such a way as to position the peg 576 in the bottommost slot in the mounting bracket 577. In this way, the frame 571 is effectively suspended above the vacuum chuck 572 (Δh). This can enable a bioprinted fibrous structure to be fully suspended for post-printing processing, such as coating the entire bioprinted fibrous structure. Regarding Figures 5I to 5K , the movement of the stage 573 can be automated such that the control system of the bioprinting system can effectively position the frame at a desired distance from the vacuum chuck (or other receiving surface) via a programmed control sequence.

[0143] Immersion bioprinting

[0144] In an embodiment, a fibrous structure can be printed into a bath containing a crosslinking agent solution, where the bath includes a member for suspending the fibers during printing. Figure 6 An exemplary drawing of such a process is shown. As shown, Figure 6 it includes a print head 602, columns 606 (only two of which are shown for clarity) included as part of the member for suspending the bioprinted fibrous structure, a surface 610, and a container 612 that is positioned on top of the surface 610 and holds the crosslinking agent solution 620. In some embodiments, as will be discussed in more detail below, the surface 610 can include the top surface of a vacuum chuck (e.g., Figures 5A to 5B the vacuum chuck 512 at ). In this way, the bioprinted fibrous structure 616 exiting from the dispensing channel 614 can be effectively crosslinked on all sides shortly after / during its dispensing. Additionally, during at least a portion of the printing process, the fibers can be suspended, in whole or at least in part, above the bottom surface 630. Printing into a bath containing a crosslinking agent solution can reduce the gravitational effects on the bioprinted fibrous structure that is suspended, in whole or at least in part, which can be used to help avoid weight-induced distortion and maintain the structural integrity of the bioprinted structure. In additional or alternative examples, printing a fibrous structure that is suspended, in whole or at least in part, into a bath containing a crosslinking agent solution can prevent the introduction of indentations / irregularities into the bioprinted fibers that may cause FBR when implanted into a subject.

[0145] In an embodiment, after generating the desired bioprinted fiber structure, the crosslinking solution may be removed. In some embodiments, removal of the crosslinking solution includes suctioning the solution out of the container or otherwise discharging (e.g., via a suitably positioned removable plug) the solution. In such an embodiment, one or more additional fluids may then be added to the container for further processing and / or storage purposes. For example, one or more washing steps may be performed, where after removal of the crosslinking solution, a wash buffer is added to the container and then removed, and this process may be repeated any number of times. In some additional or alternative embodiments, the member used to suspend the bioprinted tissue structure may be removed from the crosslinking solution to which the bioprinted fiber structure is attached. In such an example, the process of removing the member used to suspend the bioprinted structure may be done manually or may be automated. In an embodiment, once the member used to suspend the bioprinted structure has been removed from the crosslinking solution, the attached bioprinted fiber structure may optionally be further processed. In one embodiment, the member and the attached bioprinted fiber structure may be placed in another container holding another solution for further processing and / or for storage purposes. Other additional or alternative processing steps will be discussed in more detail below. Importantly, the member used to suspend the bioprinted fiber structure enables the fiber structure to be moved without compromising its structural integrity.

[0146] Go to Figure 7 , a diagram of a manufacturing platform 700 is depicted, which includes a lifting arm 702, a mounting bracket 704, a frame 706, a vacuum chuck 708, a surface 710, and a vessel 720. In Figure 7 the depicted embodiment, the vessel 720 is positioned on top of the vacuum chuck 708. In this way, the mounting bracket 704 and the frame 706 can be lowered into the solution (not specifically depicted) contained within the vessel 720. In some embodiments, the solution may include a crosslinking agent solution as discussed, and optionally, a fiber structure may be printed onto the frame 706 while submerging the frame in the crosslinking agent solution. In additional or alternative embodiments, the bioprinted fiber structure does not necessarily need to be printed into the solution, but rather the bioprinted fiber structure attached to the frame may be lowered into the solution contained within the vessel after printing. The solution may be, for example, a crosslinking agent solution, a washing solution, a storage solution, or in some embodiments, a dip coating solution (discussed in more detail below).

[0147] In some embodiments, by placing the vessel (e.g., Figure 7 the vessel 720 at ) on top of the vacuum chuck (e.g., Figure 7 the vacuum chuck 708 at ), the frame (e.g., Figure 7at 706), the vessel can then be removed from the top of the vacuum chuck, and the frame can then be lowered closely adjacent to the top of the vacuum chuck in order to rely on the associated vacuum to remove excess fluid associated with one or more of the bioprinted fiber structure, the frame, and / or the mounting bracket. This process can be repeated any number of times as needed.

[0148] Conformal coating

[0149] As discussed herein, the coating process can add one or more additional outer layers to the bioprinted fiber structure. Advantageously, the present invention enables the entire printed fiber structure (including its bottom surface) to be uniformly coated one or more times. In an embodiment, these additional one or more outer layers can impart anti-FBR properties and / or increased stability to the bioprinted fiber structure.

[0150] Now turning to Figure 8 , an exemplary process flow for adding a conformal coating to the bioprinted fiber structure of the present disclosure is depicted. In Figure 8 's exemplary process flow, the bioprinted fibers forming the fiber structure are produced in such a way that the fibers are exposed to a sheath fluid containing a crosslinking agent during printing. It should be understood that the bioprinted fiber structure discussed in the context of Figure 8 can include a mesh / lattice structure produced via a member for suspending the bioprinted fiber structure as disclosed herein (e.g., a frame as depicted in Figures 2A to 2D ).

[0151] Figure 8 Step (1) at includes printing the fiber structure. In this example, the fibers constituting the fiber structure include a core 802 and a sheath layer 804 surrounding the core, and the sheath layer is in turn surrounded by a sheath fluid 806 containing a crosslinking agent during printing. In an embodiment, the sheath layer 804 and optionally the core 802 include a crosslinkable material. In an embodiment, the core 802 is solid and optionally further includes at least one biological material (e.g., cells). Although depicted as one sheath layer, bioprinted fibers including more than one shell layer are also within the scope of the present disclosure. When printing the fiber structure onto a member for suspension, the sheath fluid is removed, for example, via flowing through a porous receiving surface (not shown) as discussed above. After printing, optional step (2) includes immersing the entire fiber structure 810 in a crosslinking solution 812 to facilitate / continue the uniform crosslinking of the entire printed fiber structure. Although not explicitly stated, in other additional or alternative embodiments, the entire fiber structure can be washed with the crosslinking solution, for example, by dispensing the crosslinking solution onto the fiber structure while maintaining suspension via a member for suspension (e.g., a frame as depicted in Figures 2A to 2D ).

[0152] Step (3) is divided into two sub - steps (3a) and (3b). Step (3a) includes coating the entire fiber structure 810 with a coating solution 816. Such coating can include, for example, dispensing the coating solution 816 onto the fiber structure while the fiber structure is suspended via a hanging member, and / or immersing the fiber structure in the coating solution 816. In an embodiment, the coating solution 816 contains a cross - linkable material (e.g., alginate). In an embodiment, the coating solution 816 contains the same material as the material that constitutes the outer shell layer 804 of the coated fiber structure 810. In an embodiment, the coating solution 816 contains a material different from the material that constitutes the outer shell layer 804 of the coated fiber structure 810. At step (3a), the residual cross - linker (e.g., Ca 2+ ) associated with the fiber structure 810 facilitates the initial cross - linking of the material in the coating solution 816 with the material that constitutes the outer shell layer 804. After achieving a conformal coating on the fiber structure 810, step (3b) includes immersing the coated fiber structure 818 in the cross - link solution 812 (or washing the coated fiber structure in other ways). In this way, the conformal coating 820 is uniformly added to the entire fiber structure, as shown in the figure.

[0153] In an embodiment, more than one coating can be added to the fiber structure in an orderly manner. For example, within the scope of the present disclosure, two, three, four, or more conformal coatings can be added to the bioprinted fiber structure in an orderly manner. By using the present invention, the fiber structure can be suspended during the application of each conformal coating, such that the entire outer surface of the resulting structure can be coated any number of times. In an embodiment, the thickness of the coating can be determined visually, for example, using a microscope.

[0154] In an embodiment, a Ca 2+ chelating agent can be applied shortly before step (3a) to remove a certain amount of Ca 2+ from the surface of the fiber structure before applying the conformal coating, so as to improve the adhesion between the fiber structure and the coating. Preferred examples of calcium chelating agents include, but are not limited to, BAPTA, EDTA, trisodium citrate, and their derivatives or analogs. A similar process can be used to add multiple conformal coatings to the bioprinted fiber structure in an orderly manner.

[0155] In an embodiment, a conformal coating can be selected based on the material composition to impart specific properties. In an example, different coatings can be composed of different materials and / or have different physical properties (e.g., different hardness levels). As an illustrative example, a first conformal coating and a second conformal coating can each be composed of alginate, but at different percentages. For example, the first conformal coating can be composed of a higher percentage of alginate (e.g., about 2%), while the second coating can be composed of a lower percentage of alginate (e.g., about 0.5%). Without being bound by theory, this approach can impart increased stability due to the higher percentage of alginate in the first coating and, at the same time, impart anti-FBR properties due to the lower percentage of alginate in the second coating (Doloff et al., Nat. Biomed. Eng. (2021); 5(10):1115 - 1130).

[0156] Accordingly, the conformal coatings implemented herein can be advantageously used to impart stability and / or impart anti-FBR properties to tissue fiber structures. With the present invention, no part of the bioprinted tissue fiber structure will lack the special properties imparted to it by one or more coatings.

[0157] In an embodiment, the coating (e.g., 820) is composed of a hydrogel material, such as a hydrogel material including one or more of alginate, chitosan, GEL-MA, poly(ethylene glycol) (PEG), poly-L-lysine (PLL), triazole, etc. In some examples, the coating is composed of a functionalized alginate, i.e., an alginate that has been chemically modified to include one or more properties, the one or more properties including but not limited to immunoprotective properties that facilitate the fabrication of the fiber structures of the present disclosure. Examples of functionalized alginates include but are not limited to methacrylated alginate, furan alginate, thiol alginate, maleimide alginate, and covalent click alginate (e.g., alginate blended with [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (DMAPS)-aldehyde (DMAPS-Ald) and / or [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (DMAPS)-hydrazide (DMAPS-Hzd)) (see U.S. Provisional Patent Application No. 63 / 192552).

[0158] In an embodiment, the coating solution comprises at least one crosslinkable material, including but not limited to hydrogels such as alginate, zwitterionic alginate, sulfobetaine methacrylate (SBMA), chitosan, poly(ethylene glycol) diacrylate (PEGDA), poly(ethylene glycol)-tetraacrylate (PEGTA), hyaluronic acid (HA), methacrylated hyaluronic acid (HAMA), collagen, methacrylated collagen (ColMA), gelatin, methacrylated gelatin (gelMA), agarose, gellan gum, fibrin (fibrinogen), poly(vinyl alcohol) (PVA), etc. or any combination thereof.

[0159] Input material

[0160] Aspects of the present invention include input 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 can be used to construct or replace tissues, such as human tissues with or without living cells. In the field of bioprinting, the term "biomaterial" is often synonymous with the term "bioink".

[0161] Input materials generally include at least one crosslinkable material, such as hydrogels, including but not limited to alginate, chitosan, PEGDA, PEGTA, hyaluronic acid (HA), HAMA, collagen, CollMA, gelatin, gelMA, agarose, gellan gum, fibrin (fibrinogen), PVA, etc. or any combination thereof, and non-hydrogels, including but not limited to PCL, poly-(d,l-lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), etc. or any combination thereof. In a preferred embodiment, the input material comprises at least one hydrogel. Non-limiting examples of hydrogels include alginate, agarose, collagen, fibrinogen, gelatin, chitosan, hyaluronic acid-based gels, or any combination thereof. A variety of synthetic hydrogels are known and can be used in embodiments of the systems and methods provided herein. For example, in some embodiments, one or more hydrogels form at least a portion of the structural basis for a 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, which may be dispersed within the hydrogel or added to the hydrogel after it is printed in a three-dimensional configuration.

[0162] In embodiments, the hydrogel can be crosslinked 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 (CaCl2), 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 an alkaline solution.

[0163] In embodiments, when exposed to a crosslinking agent material miscible with the input material, hydrogel fibers can be generated by a precipitation reaction achieved via solvent extraction from 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 the formation of precipitation-mediated hydrogel fibers include polyethylene glycol (PEG) and alginate. Crosslinking of the hydrogel will increase the hardness of the hydrogel, thereby allowing the formation of a solidified hydrogel in some embodiments.

[0164] In some embodiments, the hydrogel includes alginate. When in contact with a divalent cation, alginate forms a solidified colloidal gel (a high water content gel or hydrogel). 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+ In, Ca 2+ is the most characteristic and most commonly used to form alginate gels (Ouwerx, C. et al., Polymer Gels and Networks, 1998, 6(5): 393 - 408). Studies have shown that calcium alginate gels are formed by the cooperative binding of Ca 2+ ions on adjacent polymer chains, the so-called "egg box" model (ISP Alginates, Section 3: Algin - Manufacture and Structure, in Alginates: Products for Scientific Water Control, 2000, International Specialty Products: San Diego, pages 4 to 7). G-rich alginates tend to form thermally stable, strong, and brittle Ca gels, while M-rich alginates tend to form gels with lower thermal stability, weaker but more elastic. In some embodiments, the hydrogel includes depolymerized alginate.

[0165] In some embodiments, the hydrogel can be crosslinked using a free radical polymerization reaction to generate covalent bonds 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 persulfate (KPS) combined with N,N,N,N-tetramethylethylenediamine (TEMED) as an initiator and a catalyst, respectively. Non-limiting examples of photocrosslinkable hydrogels include: methacrylated hydrogels, such as hyaluronic acid methacrylate (HAMA), gelatin methacrylate (GEL-MA), or poly(ethylene glycol) acrylate (PEG-acrylate)-based hydrogels, which are used in cell biology due to their inertness towards cells. Polyethylene glycol diacrylate (PEG-DA) is commonly used as a scaffold in tissue engineering because the polymerization reaction occurs rapidly at room temperature, requires a low energy input, has a high water content, is elastic, and can be customized to include various biomolecules.

[0166] In an embodiment, the input material includes a non-biodegradable polymer. In an example, the input material can be a synthetic polymer, such as polyvinyl acetate (PVA). In an embodiment, the input material can include hyaluronic acid (HA).

[0167] In some embodiments, the hydrogel includes a chemically modified alginate. In an example, the chemically modified alginate includes alginate functionalized with methacrylate groups, referred to herein as "Alg-MA". In some embodiments, Alg-MA can be used for an immunoprotective shell layer via blending with zwitterionic alginate, referred to herein as "Alg-zw". Due to the dual crosslinking ability of Alg-MA, in an embodiment, Alg-MA can be first printed with Alg-zw via physical crosslinking. Upon printing, the fibers can then be further irradiated to induce covalent crosslinking across the fibers, resulting in F-F adhesion. In some embodiments, the chemically modified alginate can include thiolated alginate.

[0168] In some embodiments, one or more synthetic components can be added to the hydrogel material. The synthetic components can be used to increase fiber-to-fiber adhesion and / or in vivo stability. In an example, the material can include an acrylated zwitterionic monomer (e.g., sulfobetaine methacrylate (SBMA)) and a crosslinker (e.g., poly(ethylene glycol) diacrylate (PEGDA)). In such an example, the photo-mediated crosslinking of the zwitterionic monomer with PEGDA can render the resulting crosslinked polymer matrix superhydrophilic and thus less prone to generating a foreign body reaction (FBR) (see U.S. Provisional Patent Application No. 63 / 192552, the content of which is hereby expressly incorporated by reference in its entirety).

[0169] In some embodiments, the hydrogel material can 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") and zwitterionic monomers and hydrazide motifs (e.g., DMAPS-hydrazide, referred to herein as "DMAPS-Hzd") can be used (see U.S. Provisional Patent Application No. 63 / 192552). Aldehydes readily react with hydrazides to form covalently crosslinked hydrogels. Due to the presence of zwitterionic monomers in the polymer backbone, these polymers can exhibit low protein-binding properties. In an embodiment, one of these polymers can be blended with alginate in the shell. After printing, the structure can be immersed in a solution containing counter components, which will in turn produce covalent crosslinking bridges between the fibers, resulting in F-F adhesion.

[0170] In an embodiment, the input material includes microparticles, and as used herein, "microparticles" refers to immiscible particles in the range of about 0.1 μm to about 100 μm, which are typically composed of polymers, metals, or other inorganic materials. They can be symmetric (e.g., spherical, cubic, etc.), but this is not necessary. Microparticles with an aspect ratio of 2:1 or greater can be considered micro-rods or microfibers.

[0171] The input material according to embodiments herein can include any of a variety of natural or synthetic polymers that support the viability of living cells, including, 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, i.e., conducive to cell growth, differentiation, and communication. In certain embodiments, the input material includes one or more physiological matrix materials or combinations thereof. "Physiological matrix materials" refers to biomaterials found in natural mammalian tissues. Non-limiting examples of such physiological matrix materials include: fibronectin, thrombospondin, glycosaminoglycans (GAGs) (e.g., hyaluronic acid, chondroitin 6-sulfate, dermatan sulfate, chondroitin 4-sulfate, or keratin sulfate), deoxyribonucleic acid (DNA), adhesive glycoproteins, and collagens (e.g., collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, or collagen XVIII).

[0172] Collagen provides tensile strength to most tissues, and multiple collagen fibrils with a diameter of about 100 nm combine to generate strong coiled-coil fibers with a diameter of about 10 μm. The biomechanical function of certain tissue architectures is conferred via the oriented arrangement of collagen fibers. In some embodiments, the input material includes collagen fibrils. An input material including collagen fibrils can be used to generate a fibrous structure that forms a tissue architecture. By regulating the diameter of the fibrous structure, the orientation of the collagen fibrils can be controlled, thereby guiding the polymerization of the collagen fibrils in a desired manner.

[0173] For example, previous studies have shown that microfluidic channels of different diameters can direct the polymerization of collagen fibrils to form fibers oriented along the channel length, but only when the channel diameter is 100 μm or smaller (Lee et al., 2006). Primary endothelial cells grown in these oriented matrices were shown to align in the direction of the collagen fibers. In another study, Martinez et al. demonstrated that 500-μm channels within a beaded cellulose scaffold could direct the alignment of collagen and cells (Martinez et al., 2012). By regulating the fiber diameter, the orientation of the collagen fibers within the fibrous structure can be controlled. Thus, the fibrous structure and the collagen fibers within it can be patterned to produce a tissue architecture with a desired collagen fiber arrangement, which is essential for conferring the desired biomechanical properties on 3D printed structures.

[0174] Additional fluid

[0175] Aspects of the present invention include one or more buffer solutions. A buffer solution according to an embodiment of the present invention can be miscible with an input material (e.g., a hydrogel) and does not crosslink the input material. In some embodiments, the buffer solution comprises an aqueous solvent. Non-limiting examples of buffer solutions include polyvinyl alcohol, water, glycerol, propylene glycol, sucrose, gelatin, or any combination thereof.

[0176] The viscosity of a buffer solution according to an embodiment of the present invention can range from about 1 mPa·s to about 5,000 mPa·s, such as about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,250, 3,500, 3,750, 4,000, 4,250, 4,500, or 4,750 mPa·s. In some embodiments, the viscosity of the buffer solution can be adjusted such that it matches the viscosity of one or more input materials.

[0177] Aspects of the present invention include one or more sheath fluids. The sheath fluid according to an embodiment of the present invention can be used at least partially to encapsulate or "wrap" the input material dispensed from the dispensing channel. In some embodiments, the sheath fluid comprises an aqueous solvent. Non-limiting examples of the sheath fluid include polyvinyl alcohol, water, glycerol, propylene glycol, sucrose, gelatin, or any combination thereof. The viscosity of the sheath fluid according to an embodiment of the present invention can range from about 1 mPa·s to about 5,000 mPa·s, such as about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,250, 3,500, 3,750, 4,000, 4,250, 4,500, or 4,750 mPa·s. In some embodiments, the viscosity of the sheath fluid can be adjusted to match the viscosity of one or more input materials.

[0178] In some embodiments, the sheath fluid comprises a chemical crosslinking agent. In some embodiments, the chemical crosslinking agent comprises a divalent cation. Non-limiting examples of the divalent cation 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 the divalent cation in the sheath fluid ranges from about 80 mM to about 140 mM, such as about 90, 100, 110, 120, or 130 mM.

[0179] Cell population

[0180] In an embodiment, the cell population is selected from the group consisting of, or consists of, a single cell suspension, cell aggregates, cell spheroids, cell organoids, or any combination thereof. The input material according to an embodiment of the present invention can incorporate any mammalian cell type, including but not limited to stem cells (e.g., embryonic stem cells, adult stem cells, induced pluripotent stem cells), germ cells, endodermal cells (e.g., lung, liver, pancreas, gastrointestinal, or urogenital tract cells), mesodermal cells (e.g., kidney, bone, muscle, endothelial cells, or heart cells), and ectodermal cells (skin, nervous system, pituitary, or eye cells), stem cell-derived cells, or any combination thereof.

[0181] For example, the input material may include cells from endocrine and exocrine glands, including the pancreas (alpha, beta, delta, epsilon, gamma), liver (hepatocytes, Kupffer, stellate, sinusoidal cells), thyroid (follicular cells), pineal gland (pinealocytes), pituitary gland (somatotrophs, lactotrophs, gonadotrophs, corticotrophs, and thyrotrophs), thymus (thymocytes, thymic epithelial cells, thymic stromal cells), adrenal gland (cortical cells, chromaffin cells), ovary (granulosa cells), testis (Leydig cells), gastrointestinal tract (enteroendocrine cells - intestine, stomach, pancreas), fibroblasts, chondrocytes, meniscal 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.

[0182] The cells can be obtained from a donor (allogeneic), from a different species than the recipient (xenogeneic), or from the recipient (autologous). Specifically, in embodiments, the cells can be obtained from a suitable donor such as a human or an animal, or from the subject into whom the cells are to be implanted. Mammalian species include, but are not limited to, humans, monkeys, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, and rats. In one embodiment, the cells are human cells. In other embodiments, the cells can be derived from an animal, such as a dog, cat, horse, monkey, or any other mammal.

[0183] In some embodiments, at least one biomaterial includes a cell population that expresses / secretes one or more endogenous bioactive agents (e.g., insulin, glucagon, ghrelin, pancreatic polypeptide, factor VII, factor VIII, factor IX, alpha-1 antitrypsin, angiogenic factors, growth factors, hormones, antibodies, enzymes, proteins, exosomes, etc.). The endogenous bioactive agents discussed herein include those agents that are naturally produced by the cells in a biological environment (e.g., insulin release in response to elevated glucose concentration). The endogenous bioactive agents can constitute therapeutic agents in the context of the present disclosure.

[0184] In some embodiments, the input material may include genetically engineered cells that secrete a specific factor. As discussed above, the cell population in embodiments may include engineered cells (e.g., genetically engineered cells) that secrete a specific factor, which is within the scope of the present disclosure. The cells can also be from an established cell culture line, or can be cells that have been genetically engineered and / or manipulated to achieve a desired genotype or phenotype. In some embodiments, tissue pieces can also be used, which can provide multiple different cell types within the same structure.

[0185] Genetic engineering techniques applicable to the present disclosure may include, but are not limited to: recombinant DNA (rDNA) technology (Stryjewska et al., Pharmacologial Reports. 2013; 65:1075); cell engineering based on the use of targeted nucleases (e.g., meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease Cas9 (CRISPR-Cas9), etc.) (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 engineering based on site-specific recombination using a recombinase system (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. In some embodiments, some combinations of the techniques for cell engineering mentioned above may be used.

[0186] The present disclosure encompasses engineered cells capable of producing one or more therapeutic agents, said 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 antimicrobial 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.

[0187] In embodiments, the cells of the present disclosure can be modified to include at least one mechanism for providing local immunosuppression at the site of transplantation, e.g., in the tissue fibers of the present disclosure, when transplanted into an allogeneic host. In an example, one or more cells can comprise a set of transgenes, each encoding a cytoplasmic, membrane-bound, or locally acting gene product, and whose functions can include, but are not limited to, reducing the activation and function of antigen-presenting cells; reducing graft-attacking leukocyte activity or cytolytic function; reducing the cytolytic function of macrophages and the phagocytosis of allograft cells; inducing apoptosis in leukocytes attacking the graft; reducing local inflammatory proteins; and preventing leukocyte-mediated apoptosis (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).

[0188] In embodiments, the cells of the present disclosure can be modified in a manner that exerts control over cell proliferation. As an example, the cells can be genetically modified at a cell division locus (CDL) to include a negative selectable marker and / or an inducer-based gene expression system, whereby the proliferation of the genetically modified cells can be controlled by the addition or removal of an appropriate inducer (WO2016 / 141480; Liang et al., Nature. 2018; 563(7733):701-704).

[0189] Appropriate 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 typically include essential nutrients and optional additional elements such as growth factors, salts, minerals, vitamins, etc., which can be selected according to the cell type being cultured. Specific components can be chosen to enhance cell growth, differentiation, secretion of specific proteins, etc. Generally, standard growth media include Dulbecco's Modified Eagle Medium, low glucose (DMEM), containing 110 mg / L pyruvate and glutamine, supplemented with 10% to 20% fetal bovine serum (FBS) or calf serum and 100 U / ml penicillin, which is as suitable as various other standard media well known to those skilled in the art. Growth conditions will vary depending on the mammalian cell type used and the desired tissue.

[0190] In some embodiments, cell type - specific reagents can be advantageously used in the subject input material for the corresponding cell type. For example, the extracellular matrix (“ECM”) can be directly extracted from the tissue of interest and then solubilized and incorporated into the input material to generate a tissue - specific input material for printing tissue. Such ECM can be readily obtained from patient samples and / or can be commercially available from suppliers such as zPredictA (rBone TM , available at zpredicta.com / home / products).

[0191] Printing system

[0192] Although bioprinting systems vary, they generally include at least one reservoir that contains 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 needles, syringe, nozzle, etc.). The dispensing orifice can have a suitable shape, such as circular, square, oval, oblong, rectangular, etc. In some examples, the bioprinting system includes multiple reservoirs and / or can include a component for selecting a reservoir to be used for bioprinting from the multiple reservoirs. A bioprinting system that includes one or more reservoirs can be advantageous in continuous or substantially continuous bioprinting applications. The volumes of the reservoirs vary, for example, from about 100 pl to about 1 L or greater, including any intermediate values therebetween, such as about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mL, or such as about 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or 950 mL.

[0193] In an example, the interior of the reservoir can be composed of a material that resists cell attachment. In an example, at least the interior of the reservoir can be composed of a biocompatible material. The reservoir can be compatible with bioprinting involving extrusion of a semi-solid or solid bioink or support material through one or more dispensing orifices. The reservoir can be compatible with bioprinting involving dispensing of a liquid or semi-solid cell solution, cell suspension, or cell concentrate through one or more dispensing orifices. The reservoir can be compatible with non-continuous bioprinting, continuous and / or substantially continuous bioprinting. The reservoir can include, but is not limited to, a capillary, micropipette, syringe, needle, bottle, basin, receptacle, etc. Many inner diameters are suitable for substantially circular or cylindrical reservoirs.

[0194] In some examples, the reservoir of the bioprinting system can be primed. For example, priming the reservoir can improve the accuracy of the dispensing and / or deposition process by, for example, pressing and advancing the contents of the reservoir until the material to be dispensed (e.g., bioink) is in contact with the dispensing orifice.

[0195] In a preferred embodiment, the bioprinting system includes 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. As detailed therein, the disclosed bioprinting system and its components enable multi-material switching and thus can change the composition of one or more components of the synthetically generated tissue fibers (e.g., cell type and biomaterial composition) along the length of the fiber while printing continuously. In an embodiment, the microfluidics-based bioprinting system is RX1 TM Bioprinter (Aspect Biosystems, Vancouver, BC, Canada).

[0196] In an exemplary embodiment of a preferred bioprinting system, the system includes a printhead that includes a dispensing channel where one or more material channels and a core channel converge at the proximal end of the dispensing channel. The printhead can be configured to dispense a buffer solution and / or a sheath fluid simultaneously with one or more crosslinkable materials. In some embodiments, the printhead is configured to maintain a constant mass flow rate in the dispensing channel. In this way, the printhead 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) as well as the buffer solution and / or the sheath fluid through the dispensing channel. In the use of such a printhead, the input material flowing through the dispensing channel can be crosslinked internally by the fluid flowing through the core channel and / or externally by the sheath fluid flowing through a downstream sheath fluid channel, as described more specifically in WO2020 / 056517. In some embodiments, the printhead includes one or more fluid focusing chambers having a frustoconical shape and optionally one or more printhead adapters, as detailed in WO 2021 / 081672 and U.S. Provisional Patent Application No. 63 / 290595. In an embodiment, the printhead is DUO TM Microfluidic printhead, or CENTRA TM Microfluidic printhead (Aspect Biosystems, Vancouver, BC, Canada).

[0197] Other examples of bioprinting systems relevant in the context of the present disclosure include, but are not limited to, 3-D (EnvisionTEC Inc., Dearborn, MI, USA), NovoGen Platform( (San Diego, CA, USA), R-Gen100 and R-Gen 200 (RegenHU, Villas-Saint-Pierre, Switzerland), Bioprinter Fabion and Fabion 2 (3D Bioprinting Solutions, Moscow, Russia), Basic, 200 / 400 / 500 (Advanced Solutions, Louisville, KY, USA), BIO X TM , BIO X6 TM , INKREDIBLE+ TM (CellINK, Boston, MA, USA), Ourobotics Revolution (Ourobotics, Cork, Ireland), BioScaffolder 2.1 (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, South Korea).

[0198] As discussed above, a bioprinting system typically dispenses a bioprinting material onto a receiving surface. As discussed herein, a bioprinted fibrous structure is printed using a member for suspending the bioprinted fibrous structure during one or more of printing, patterning, and / or processing. In an embodiment, the fibrous structure can be printed onto a receiving surface (e.g., Figures 5A to 5B the mesh 514 at Figures 5A to 5B ) by using a member for suspending the structure (e.g., the frame 508 at

[0199] The bioprinting systems disclosed herein can be trimmed or otherwise modified to provide a surface capable of accommodating components for suspending bioprinted tissue constructs as disclosed herein. In an embodiment, the surface can be modified or otherwise configured in a manner so as to be operably coupled to one or more vessels (e.g., containers, well plates, etc.). By way of example and not limitation, a container can include a vessel capable of holding a solution (such as a crosslinker solution or a dip coating solution as described herein). In an embodiment, the size and shape of such a container can be such that an entire component for suspending bioprinted fibers can be loaded therein, and the liquid held in the container can fully immerse the component for suspending bioprinted fibers and, in turn, fully immerse the bioprinted fiber structure attached to the component.

[0200] In an embodiment, the receiving surface is disposable. In an embodiment, the receiving surface can be effectively sterilized. In an embodiment, the receiving surface includes a solid material, a semi-solid material, or some combination thereof. In an embodiment, the receiving surface is porous. In an embodiment, the receiving surface includes glass, coated glass, plastic, coated plastic, metal, metal alloy, mesh, grating, or a combination thereof.

[0201] In some embodiments, the bioprinting system includes a fluid removal component for removing excess fluid (e.g., excess sheath fluid and / or excess buffer solution) from the receiving surface and / or from the surface of the dispensed tissue fiber structure. During printing, excess fluid has the potential to accumulate or "pool" on the receiving surface or on the surface of the dispensed tissue fiber structure, and in some instances, such pooling can interfere with one or more aspects of the deposition process. For example, in the context of the present disclosure, unwanted excess fluid can potentially reduce the ability of the fiber structure to adhere to a post (e.g., post 206 at frame 202 in FIG. 2) of a component for suspending the bioprinted fiber structure, can add an unwanted weight (which may be unevenly distributed) to the bioprinted fibers, etc., which can cause the dispensed fibers to slip from their intended positions in the 3D structure being printed. Accordingly, in some embodiments, removing excess sheath fluid from the receiving surface and / or from the surface of the dispensed fiber structure by way of the fluid removal component can improve the additive manufacturing of the three-dimensional structure. Figure 2A By pumping the fluid out of those surfaces, by allowing or facilitating the evaporation of the fluid from those surfaces, excess fluid can be removed from the receiving surface or from the surface of one or more layers of the dispensed fibers, or in embodiments where the receiving surface is porous, the excess fluid can be removed by suctioning the excess fluid through the porous surface. In some embodiments, an absorbent material (e.g., a sponge) can be used to wick excess fluid away from the receiving surface.

[0202]

[0203] ​In some embodiments, the receiving surface includes a vacuum component (e.g., Figures 5A to 5B a vacuum chuck 512 at Figures 5A to 5B ), which is configured to apply suction force from one or more vacuum sources to the receiving surface. In some embodiments, the receiving surface includes one or more vacuum channels, which are configured to apply suction force to the receiving surface. In some embodiments, the receiving surface including the vacuum component is configured to suck excess fluid from the receiving surface before, during, and / or after the printing process. In some embodiments where the receiving surface is porous, the vacuum component may be configured to apply suction force to suck excess fluid through the porous surface.

[0204] In some embodiments, the receiving surface includes one or more tubes fluidly coupled to a vacuum source, and the one or more tubes can provide suction force to remove 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 configured to further include one or more vacuum channels, each having an orifice located near (i.e., adjacent to) the dispensing orifice. When the printhead is in vacuum fluid communication, the one or more vacuum channels can direct negative pressure to the area of the receiving surface where material is being dispensed or has been dispensed from the dispensing orifice and / or a portion of the surface area of the dispensed fibrous structure, thereby sucking away excess fluid from the receiving surface and / or the surface of the dispensed fibrous structure.

[0205] In some embodiments, the bioprinter system can employ a dispensing member to control the material flow in fiber generation. For example, the bioprinter system can employ material displacement to control the material flow. In an embodiment, the dispensing member provides a force to dispense one or more materials to be dispensed. In one embodiment, the dispensing member provides pneumatic pressure to supply the force for dispensing one or more materials. In some embodiments, the bioprinter system can be configured with one or more pumps. Examples include but are not limited to gear pumps, peristaltic pumps, cam pumps, piston pumps (e.g., double piston pumps), syringe pumps, etc. In some embodiments, pumps are used to apply a force to the material contained in the reservoir to assist in moving the material towards the associated dispensing orifice.

[0206] In a preferred example, a bioprinter system used in the context of the present disclosure includes a radial pump assembly configured to minimize the internal volume in a multi-channel bioprinting system, preferably a microfluidic bioprinting system. The radial pump assembly includes a plurality of pumps positioned in a radial array on a mounting bracket, where each pump of the plurality of pumps includes a housing and a retainer for securing 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 incorporated herein by reference in its entirety.

[0207] In an embodiment, a bioprinting system related to the present disclosure may include some type of enclosure system. The enclosure system may be configured to enclose a part or all of the bioprinting system. Preferably, the enclosure system encloses at least the area where bioprinting fibers are dispensed, such as at least a receiving surface, preferably at least the receiving surface and one or more print heads, optionally encloses any dispensing member (e.g., an injection pump) configured to deliver a flowing material to a dispensing orifice, and optionally encloses one or more reservoirs. In an embodiment, such an enclosure system may be configured to adopt an open configuration in which aspects of the bioprinting system are accessible; and a closed configuration in which specific aspects of the bioprinting system are sealed off or substantially isolated from the surrounding atmosphere.

[0208] Reliance on the enclosure system enables accurate control of parameters including but not limited to temperature, humidity, O2, and CO2. Accurate control of such parameters enables increased stability and integrity of 3D bioprinted structures, and increased cell survival during and after the bioprinting process in cases where the 3D bioprinted structure includes cell material. In an embodiment, variables such as temperature, humidity, O2, and CO2 may be controlled within the enclosure system by a feedback control system (e.g., a proportional-integral-derivative (PID) controller), see, e.g., Matamoros M et al. (2020) Micromachines, 11, 999. In an embodiment, such a feedback control system may, for example, stabilize the temperature within the enclosure system to a desired temperature (within a certain error margin) and / or stabilize the humidity within the enclosure system to a desired humidity (within a certain error margin). In some additional or alternative embodiments, such a feedback control system may stabilize CO2 to a desired level (e.g., a desired ppm within a certain error margin). In some additional or alternative embodiments, such a feedback control system may stabilize O2 to a desired level (e.g., a desired ppm within a certain error margin).

[0209] Accordingly, in an embodiment, a bioprinting system related to the present disclosure may include one or more of a temperature regulating component, a humidity regulating component, an O2 regulating component, and a CO2 regulating component. In an embodiment, the temperature regulating component includes a heater (e.g., a radiant heater, a convective heater, a conductive heater, a fan heater, a heat exchanger, or any combination thereof). In an embodiment, the temperature regulating component includes a cooling element (e.g., a coolant, a cooled liquid, a Peltier cooler, a radiant cooler, a convective cooler, a conductive cooler, a fan cooler, or any combination thereof). The humidity regulating component may include, for example, a chamber (e.g., a tank) of water that can be evaporated via a piezoelectric transducer. O2 can be controlled by O2 injection. CO2 can be controlled by CO2 injection. In an embodiment, the temperature regulating component is capable of adjusting and / or maintaining the temperature within one or more of the enclosure system and / or the print head, the printer stage, the receiving surface, the flowing material, and / or the fluid (e.g., the sheath solution and / or the buffer solution). In an embodiment, the temperature regulating component is configured to adjust the temperature to a set point in the range of from about 0 °C to about 90 °C, such as about 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, or 85 °C. In an embodiment, the humidity regulating component is configured to adjust the humidity to a 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 CO2 regulating component is configured to adjust the CO2 level to a level between about 2% and about 15%, such as about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%.

[0210] In an embodiment, a bioprinting system related to the present disclosure achieves a specific geometry of the dispensed fibrous structure by moving the printer stage or surface (e.g., the receiving surface) relative to the dispensing orifice, on top of which a member for suspending the bioprinted fibrous structure is disposed. In an alternative embodiment, a bioprinting system related to the present disclosure achieves a specific geometry of the dispensed fibrous structure by moving the dispensing orifice (optionally multiple dispensing orifices) relative to the printer stage or surface (e.g., the receiving surface), on top of or above which a member for suspending the bioprinted fibrous structure is disposed. In certain embodiments, at least a portion of the bioprinting system is maintained in a sterile environment (e.g., within a biological safety cabinet (BSC)). In some embodiments, the bioprinting system is configured to be fully suitable for a sterile environment.

[0211] In some embodiments, the bioprinting system includes a 3D motorized stage (also referred to herein as a positioning unit), the 3D motorized stage including at least three arms for positioning a surface (e.g., a receiving surface) in a three-dimensional space (i.e., along the x-axis, y-axis, and z-axis of a Cartesian coordinate system) below a dispensing orifice, on top of which a member for suspending a bioprinted fibrous structure is disposed. 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), on top of or above which a member for suspending a bioprinted fibrous structure is disposed.

[0212] In some embodiments, the 3D motorized stage arms are driven respectively by corresponding motors and controlled by a programmable control processor such as a computer. In an embodiment, the surface (e.g., a receiving surface) on top of which a member for suspending a bioprinted fibrous structure is disposed can be moved along all three major axes of the Cartesian coordinate system by the 3D motorized stage, and the movement of the stage is defined using computer software. In some additional or alternative embodiments, the dispensing orifice (or orifices) can be moved along all three major axes of the Cartesian coordinate system by the 3D motorized stage, and the movement of the stage is defined using computer software.

[0213] It should be understood that the present invention is not limited solely to the described positioning system, and other positioning systems are known in the art. When dispensing material from a dispensing orifice (e.g., on a microfluidic printhead), the positioning unit moves in a software-controlled pattern to create a first layer of bioprinted fibers dispensed around a post (e.g., post 206 at Figure 2A the frame 202 at Figure 2A ). Additional layers of dispensed material are then stacked on top of each other such that the final 3D geometry of the dispensed material layers is typically a replica of the 3D geometry design provided by the software. The 3D design can be created using typical 3D CAD (computer-aided design) software or generated from a digital image, as is known in the art. Additionally, if the software-generated geometry contains information about the specific materials to be used, according to one embodiment of the present invention, specific flow material types can be assigned to different geometric locations. For example, in some embodiments, the printed 3D structure can include two or more different input materials, where each input material has different properties (e.g., each input material includes a different cell type, different cell concentration, different extracellular matrix (ECM) composition, different types of materials (e.g., different types of hydrogel materials), etc.).

[0214] In some embodiments, a bioprinting system includes a light module for optionally exposing a photocrosslinkable flowable material to light to crosslink the material. In an embodiment, the light module (e.g., an ultraviolet (UV) light module) can be integrated into the bioprinting system (e.g., integrated into its print head) or can be a stand-alone component of such a system. In some embodiments, the light module is annular. In some embodiments, the annular light module completely surrounds a transparent portion of a dispensing channel (or dispensing needle / nozzle / syringe) and / or a dispensing orifice. In some embodiments, the annular light module is positioned directly below the dispensing orifice.

[0215] In an embodiment, the annular module can be configured with a plurality of light sources to direct light inward in the direction of the central cavity of the annular light module such that when printing fibers, the light is directed circumferentially to the fibers. In an embodiment, the plurality of light sources includes at least 10 to 40, such as 15 to 35, such as 20 to 30 individual light sources. In an embodiment, the light sources include light-emitting diodes (LEDs), such as UV-LEDs. Such an annular light source and related examples of its incorporation in a bioprinting system are described in detail in U.S. Provisional Patent Application No. 63 / 290595, the content of which is incorporated herein by reference in its entirety.

[0216] Aspects of the bioprinting system include a software program configured to facilitate deposition of a subject flowable material in a specific pattern and at a specific location so as to form a specific planar or 3D structure in a certain manner, whereby the structure is deposited around members (e.g., Figure 2A the post (e.g., Figure 2A post 206 at Figures 5A to 5BPart of the manufacturing platform 500). The resulting structure can include one, two, three, four, five, or more than five layers, such as 10 layers or more, 20 layers or more, 30 layers or more, 40 layers or more, 50 layers or more, 60 layers or more, 70 layers or more, 80 layers or more, 90 layers or more, 100 layers or more, 110 layers or more, 120 layers or more, 130 layers or more, 140 layers or more, 150 layers or more, 160 layers or more, 170 layers or more, 180 layers or more, 190 layers or more, 200 layers or more. In some embodiments, the manner in which the printing system deposits the material (i.e., the location and overall deposition pattern) is defined by user input and translated into computer code. In some embodiments, the computer code includes a sequence of instructions that can be executed in the central processing unit (CPU) of a digital processing device and is written to perform a specified task. In some embodiments, the printing parameters, including but not limited to the printed fiber size, pump speed, movement speed of the positioning unit, and crosslinker strength or concentration, are defined by user input and translated into computer code. In some embodiments, the printing parameters are not directly defined by user input but are derived from other parameters and conditions through computer code.

[0217] Aspects of the present invention include a method for manufacturing a fibrous structure, the method comprising: a computer module receiving an input of a visual representation of a desired tissue construct; the computer module generating a series of commands, wherein the commands are based on the visual representation and 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.

[0218] In some embodiments, the manner in which the bioprinting system deposits the input material (i.e., the location and overall deposition pattern) is defined by user input and translated into computer code. In some embodiments, the devices, systems, and methods disclosed herein further include a non-transitory computer-readable storage medium or a storage medium encoded with computer-readable program code. In some embodiments, the computer-readable storage medium is a tangible component of the bioprinting system (or its components) or a computer connected to the bioprinting system (or its components). In some embodiments, the computer-readable storage medium is optionally removable from the digital processing device. In some embodiments, by way of non-limiting example, the computer-readable storage medium includes CD-ROMs, DVDs, flash memory devices, solid-state memories, disk drives, tape drives, optical disc drives, cloud computing systems and / or services, etc. In some cases, the programs and instructions are permanently, substantially permanently, semi-permanently, or non-transitorily encoded on the storage medium.

[0219] In some embodiments, the devices, systems, and methods described herein include software, server, and database modules. In some embodiments, a "computer module" is a software component (including a code segment) that interacts with a larger computing system. In some embodiments, a software module (or program module) is in the form of one or more files and generally disposes of a particular task within a computing system.

[0220] In some embodiments, a module is included within one or more software systems. In some embodiments, a module is integrated with one or more other modules into one or more software systems. A computer module is optionally a stand-alone code segment or optionally code that is not separately identifiable. In some embodiments, a module is within a single application. In other embodiments, a module is 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 more than one location. A computer module according to an embodiment of the present invention allows an end user to use a computer to perform one or more aspects of the methods described herein.

[0221] In some embodiments, a computer module includes a graphical user interface (GUI). As used herein, a "graphical user interface" means a user environment that uses pictorial and textual representations of inputs and outputs of an application and hierarchical or other data structures storing information. In some embodiments, a computer module includes a display screen. In additional embodiments, a computer module presents a two-dimensional GUI via the display screen. In some embodiments, a computer module presents a three-dimensional GUI via the display screen, such as a virtual reality environment. In some embodiments, the display screen is a touch screen and presents an interactive GUI.

[0222] Quality control system

[0223] Quality assurance for 3D bioprinting of fibers via a printing system (such as those systems disclosed herein) is crucial for reproducible biofiber fabrication, functionality, and regulatory approval for any translational application. Accordingly, the bioprinting systems of the present disclosure may incorporate one or more of the quality control systems discussed below. In an embodiment, the quality control system includes one or more cameras. In embodiments where at least a portion of the dispensing orifice (and / or dispensing channel / needle / syringe / nozzle leading to the dispensing orifice) and / or other aspects of the system (e.g., a microfluidic printhead) are transparent, one or more cameras may be used to image the material stream to be dispensed for the purpose of detecting blockages or other anomalies in the material stream.

[0224] For example, in an embodiment, the microfluidic printhead of the present disclosure (e.g., similar or substantially the same as the DUO TM microfluidic printhead or CENTRA TM microfluidic printhead (Aspect Biosystems, Vancouver, BC, Canada)) includes a transparent dispensing channel. In such embodiments, the camera system may include a first camera positioned at a first angle relative to the transparent dispensing channel and a second camera positioned at a different second angle relative to the transparent dispensing channel. In an embodiment, the two cameras may be oriented at an angle of approximately 90° relative to each other. The first camera and the second camera may form part of a machine learning-based system capable of identifying one or more deviations from user-established material flow parameters in the material flow. For example, such a system may be capable of monitoring fiber concentricity, various fiber properties, the presence or absence of blockages, air bubbles, etc., and may also be capable of controlling one or more parameters (e.g., valve opening / closing, 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 incorporated herein by reference in its entirety.

[0225] In an embodiment, one or more additional or alternative cameras may be included as part of the bio-printing system of the present disclosure. In one such embodiment, the camera may be aimed at a member for suspending the bio-printed fiber structure (e.g., the frame 202 at FIG. 2) so as to be able to image the bio-printed fiber structure during the printing of the bio-printed fiber structure and monitor one or more properties associated with the fibers during printing. Such properties may include, but are not limited to, the presence and / or absence of leading and / or trailing fibers and the shape fidelity of the printed fibers and / or the 3D structure formed by the printed fibers.

[0226] In an embodiment, an annular light module, such as the annular light module discussed above, may form part of a quality control system because the annular light module can be used to ensure the crosslinking uniformity of fibers composed of photo-crosslinkable materials during fiber printing.

[0227] The present disclosure also recognizes that, in the context of the present disclosure, the use of pneumatic valves may be advantageous in reducing or avoiding inaccuracies or distortions in a desired 3D structure. For example, a printing system incorporating the present disclosure that relies on one or more pneumatic valves can reduce or avoid inaccurate starts and / or stops of the printing process, which otherwise could result in the formation of leading and / or trailing fibers, which could in turn distort the dimensions of the printed fibers and 3D structure. In fibers composed of more than one type of material along the fiber length, relying on pneumatic valves can also be advantageous for accurately switching the material type from one type to another without forming a leading end and / or a trailing end. Such accurate switching enables effective compartmentalization of different segments along the fiber length. For example, a fiber can include a first segment composed of a first type of hydrogel, a second segment composed of a second type of hydrogel (where the second segment optionally includes cells), and a third segment composed of yet another type of hydrogel. This example is intended to be illustrative, and one of ordinary skill in the art can select the material type, and optionally the cell type (or lack thereof), for each compartment along the fiber length based on the needs of a particular application.

[0228] The embodiments and examples described above are merely illustrative and not restrictive. Those skilled in the art will recognize or be able to determine many equivalents of specific compounds, materials, and procedures using only routine experimentation. All such equivalents are considered to be within the scope of the appended claims and are covered by the appended claims.

[0229] Manufacturing method

[0230] Aspects of the present invention include methods of printing linear fiber structures, planar structures including one or more fiber structures, or three-dimensional (3D) structures including two or more layers of planar structures. The ways of printing the first layer and optionally the second layer, third layer, etc. (e.g., 200 layers or more are within the scope of the present disclosure) were described in detail above with reference to Figures 3 to 4 and Figure 6 In some embodiments, a linear fiber structure can be produced by winding a bioprinting fiber around a first post (e.g., Figure 2A post 206) one or more times, then continuing to print the fiber and winding the fiber around a second post one or more times, preferably where the first post and the second post are on opposite sides of a member (e.g., Figure 2A frame 202) for suspending the bioprinting fiber structure. In some embodiments, a method first includes providing a design for a linear, planar, or 3D structure to be printed. The design can be created using commercially available CAD software. In some embodiments, the design includes information about the specific materials to be assigned to specific locations in the structure to be printed (e.g., for a heterogeneous structure including multiple materials).

[0231] In an embodiment, a method includes dispensing a bioprinted fiber into a crosslinker bath, where the fiber is printed via a member for suspending the bioprinted fiber (e.g., the frame 202 at FIG. 2), and where the member and in turn the resulting fiber are immersed in the crosslinker bath. In some embodiments of such a method, the bioprinted fiber may be additionally crosslinked before being dispensed into the crosslinker bath. For example, the bioprinted fiber may be crosslinked via a sheath fluid containing a crosslinker or via photocrosslinking before being introduced into the crosslinker bath. In other embodiments, the bioprinted fiber may not be crosslinked before being dispensed into the crosslinker bath. In still some other embodiments, the bioprinted fiber may be produced in such a way that the fiber is crosslinked (e.g., exposed to a sheath fluid containing a crosslinker and / or photocrosslinked) under conditions where the member for suspending the bioprinted fiber is not positioned in the crosslinker bath before the fiber is dispensed onto the member for suspending the bioprinted fiber. In some embodiments, after printing the crosslinked fiber, the member for suspension and the correspondingly attached bioprinted fiber may be immersed in the crosslinker bath. The above was discussed for Figure 8 such an exemplary process flow.

[0232] In an embodiment, a method includes conformally 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 a bioprinted fiber (e.g., a 3D structure) that has been printed using a member for suspension as disclosed herein (e.g., Figure 2A the frame 202 at ), and applying a desired coating material while the fiber structure is suspended above a receiving surface via the member for suspension. In this way, a conformal coating including the coating material can be added to the suspended bioprinted fiber. In an embodiment, a method includes producing a crosslinked bioprinted fiber structure (e.g., a 3D structure) in any of the ways described above, and then immersing the entire fiber structure in a coating solution while the fiber structure remains attached to the member for suspending the bioprinted fiber structure.

[0233] In other additional or alternative embodiments, the coating solution (e.g., via a print head, such as a microfluidic print head) may be applied to the structure while the fiber structure remains attached to the member for suspending the fiber structure, and where the fiber structure is suspended away from the receiving surface. As discussed herein, in an embodiment, multiple (e.g., at least 2, 3, 4, 5, 6, or more) coatings may be applied to the bioprinted fiber.

[0234] In some embodiments of the methods described herein, the solution in which the bioprinted fibers are immersed (e.g., crosslinker solution, buffer solution, coating solution) can be removed via aspiration or via drainage (e.g., through one or more pluggable orifices). In this way, a single vessel can be used to immerse the bioprinted fibers in different solutions. For example, the bioprinted fibers attached to a member for suspension (e.g., Figure 2A the frame 202 at

[0235] In some embodiments of the methods described herein, to facilitate removal of one solution (e.g., crosslinker solution) in which a bioprinted fiber structure is immersed and then, optionally, subsequently immersing the fiber structure in another solution (e.g., coating solution) or otherwise processing (e.g., depositing a coating solution onto the fiber structure), the member for suspension (e.g., Figure 2A the frame 202 at

[0236] In an embodiment, a manufacturing platform such as the manufacturing platform discussed above with respect to Figure 7 can be used to alternately immerse and remove the fiber structure attached to a member for suspending the fiber structure by manipulating a lifting arm (e.g., Figure 7 the lifting arm 702 at Figure 2B In other embodiments, the user can simply rely on, for example, a handle (e.g.,

[0237] In an embodiment, a method for a bioprinted fiber structure includes surrounding a member for suspending the bioprinted fiber structure (e.g., Figure 2ATwo or more columns associated with the frame 202) at print at least one hydrogel fiber of a continuous length, the columns being, for example, 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 even 100 or more columns (e.g., Figure 2A the columns 206) at . In an embodiment, the member is included as part of a manufacturing platform as disclosed herein and / or is included as part of a bioprinting system as disclosed herein. In an embodiment, the method is used to produce a planar structure (i.e., one layer). In an embodiment, the method is used to produce a 3D structure (i.e., two or more layers). In an embodiment, the method further includes conformally coating the entire bioprinted fiber structure such that its upper and lower sides as well as its side walls are uniformly coated. In an embodiment, the method further includes conformally coating the entire bioprinted fiber structure multiple times (e.g., at least 2 times). Each conformal coating may comprise the same or different coating material compositions.

[0238] In an embodiment, a method for a bioprinted fiber structure includes printing a fiber structure composed of a core and at least one shell layer surrounding the core. In an embodiment, a coating material is applied to the entire fiber structure to produce a uniform conformal coating of the structure. The conformal coating may impart stability to the fiber structure, and / or may impart to the structure properties that optimize the interface between the fiber structure and the host, such as anti-FBR properties, promoting vascularization, etc. In an embodiment, the coating may be softer than the outermost outer shell. In an embodiment, the core, outer shell, and conformal coating may comprise from about 0.1% to about 4% alginate. In an embodiment, the fiber structure may include a core having from about 0.75% to 1.5% alginate, an outer shell having from about 1.5% to 2.5% alginate, and a conformal coating having from about 0.2% to 2.5% (such as 0.2% to 0.75%) alginate.

[0239] In an embodiment where two coatings are applied, within the scope of the present disclosure, the innermost coating may have a greater hardness than the outermost coating. In an embodiment, the core, outer shell, and conformal coating may comprise from about 0.1% to about 4% alginate. In an embodiment, the fiber structure may include a core having from about 0.75% to 1.5% alginate, an outer shell having from about 1.5% to 2.5% alginate, an inner conformal coating having from about 1.5% to 2.5% alginate, and an outer conformal coating having from about 0.2% to 2.5% (such as 0.2% to 0.75%) alginate. Such examples are intended to be illustrative.

[0240] In an embodiment, a bioprinted fiber structure made by the methods disclosed herein can be segmented / divided along at least a portion of the length of the fibers (preferably continuous fibers) that make up the bioprinted fiber structure. Details of generating a segmented / divided bioprinted fiber structure are described in U.S. Provisional Patent Application No. 63 / 192552, the content of which is hereby incorporated by reference in its entirety. In an embodiment, the fiber consists of a core and an outer layer, referred to herein as a core-shell fiber, wherein the core and / or the outer layer is segmented / divided along at least a portion of the fiber length. In an embodiment, the fiber consists of a core, at least one inner shell layer, and at least one outer shell layer, referred to herein as an annular fiber, wherein any one or more of the core, the inner shell layer, and / or the outer shell layer is segmented / divided along at least a portion of the fiber length. In an embodiment, one or more segments / compartments of the core and / or the shell layer can consist of a biomaterial (e.g., cells).

[0241] The compartment size can be a function of one or more variables, including but not limited to tissue fiber size (e.g., length and / or diameter), fiber type (e.g., core-shell fiber, annular fiber), type of material used in the tissue fiber generation process, whether the generated fiber structure is coated one or more times, etc. In some embodiments, the fiber can consist of at least two segments / compartments including a biomaterial, wherein other segments flanking the at least two segments / compartments do not contain a biomaterial. For example, in the case of a core-shell fiber, at least two segments containing a biomaterial can be included within the core. In another example, in the case of an annular fiber, at least two segments containing a biomaterial can be included within the first shell layer. In an embodiment, the segments including a biomaterial can have a longer length than the segments lacking a biomaterial. In an embodiment, the segments including a biomaterial can be substantially similar in length compared to the segments lacking a biomaterial. In an embodiment, the segments including a biomaterial can have a smaller length than the segments lacking a biomaterial. In an embodiment, the segments including a biomaterial for a particular tissue fiber do not have to be of the same approximate length, but different segments can include different lengths. In an embodiment, the segments lacking a biomaterial for a particular tissue fiber do not have to be of the same approximate length, but different segments can include different lengths. In some embodiments, the spacing between compartments / segments including a biomaterial (e.g., cells) in the tissue fibers of the present disclosure can be between 1-5 mm, e.g., spaced 1 mm, 2 mm, 3 mm, 4 mm, or 5 mm apart.

[0242] A section / compartment composed of a biomaterial may include, for example, cells of a specific density. In an embodiment, the density between compartments may be the same. In an embodiment, the density between compartments may be different. In an embodiment, the biomaterials between compartments may be the same or different. In an embodiment, the density of the biomaterial may be selected based on one or more of a specific application (e.g., treatment of a specific disease / ailment), cell viability determinants, the material including the biomaterial (e.g., a biocompatible material), etc. As an example, the biologic may include islets of Langerhans. Other biomaterials (e.g., hepatocytes) may be used in the tissue fibers of the present disclosure at the same or different densities.

[0243] In an embodiment, one or more sections / compartments including a biomaterial may be flanked by a section including, for example, a material having immunoprotective properties. By way of illustration and not limitation, an immunoprotective hydrogel material may include, for example, functionalized alginate, including but not limited to methacrylated alginate, furan alginate, thiol alginate, maleimide alginate, and covalently clickable alginate (e.g., alginate blended with DMAPS - Alg and / or DMAPS - Hzd). For example, in the case of a core - shell fiber, where the core includes two or more sections containing a biomaterial, the two or more sections may be flanked by other sections including an immunoprotective material as disclosed herein. In other embodiments, without departing from the scope of the present disclosure, two or more sections including a biomaterial may be flanked by other sections that do not include, for example, an immunoprotective material. Similar logic applies to the annular fibers of the present disclosure. For example, an annular fiber may be composed of two or more sections / compartments composed of a biomaterial, where each of the two or more sections / compartments may be flanked by a section incorporating, for example, an immunoprotective material of the present disclosure. In other embodiments, without departing from the scope of the present disclosure, two or more sections including a biomaterial may be flanked by sections that do not include, for example, an immunoprotective material.

[0244] Method of Use

[0245] Aspects of the method include providing one or more input materials to be dispensed through a dispensing orifice. In some embodiments, one or more cell types are compatible with the input material and are optionally dispensed within the input material. In some embodiments, a sheath fluid is used as a lubricant for lubricating the movement of the input material (e.g., within a microfluidic printhead). In some embodiments, the sheath fluid contains a cross - linker for curing at least a portion of the hydrogel before or during dispensing from the dispensing orifice. In some embodiments, the cross - linker may be included in the input material, such as the input material corresponding to the core of the fiber of the present disclosure.

[0246] Aspects of the method include transmitting a design to a 3D printer. In some embodiments, the communication can be implemented, for example, by a programmable control processor. In some embodiments, the methods include: controlling the relative positioning of an dispensing orifice and a receiving surface in three-dimensional space, and simultaneously and individually or in combination dispensing an input material and, in some embodiments, a sheath fluid from the dispensing orifice. In some embodiments, the dispensed materials are dispensed coaxially such that the sheath fluid encapsulates the input material. This coaxial arrangement allows a crosslinking agent in the sheath fluid to cure the input material, thereby producing a cured fiber structure that is then dispensed through the dispensing orifice.

[0247] In some embodiments, a method includes: depositing a first layer of the dispensed fiber structure via a member for suspending bioprinted tissue fibers (e.g., the frame 202 at FIG. 2), the first layer including an arrangement of the fiber structure specified by the design, and iteratively repeating the depositing step; depositing subsequent fiber structures onto the first layer and subsequent layers, thereby depositing the dispensed fiber structures layer by layer in a geometric arrangement specified by the design to produce a 3D structure.

[0248] In some embodiments, a plurality of input materials, such as a plurality of hydrogels, are deposited in a controlled sequence, where at least some include one or more cell types, thereby allowing a controlled arrangement of the input materials and cell types to be deposited in a geometric arrangement specified by the design.

[0249] In some embodiments, a method includes removing excess fluid from the receiving surface and / or from the surface of the dispensed fiber structure. For example, the step of removing excess fluid can be continuously performed throughout the printing process, thereby removing excess fluid that might otherwise interfere with the layering of the dispensed fiber structures in the geometric arrangement provided by the design. Alternatively, the step of removing excess fluid can be intermittently performed in sequence or simultaneously with one or more depositing steps throughout the printing process. In some embodiments, the removal of excess fluid is achieved by pumping the fluid from the receiving surface and / or from the surface of the dispensed fiber structure. In some embodiments, the removal of excess fluid is achieved by pumping excess fluid via the receiving surface or other surface, the surface including pores sized to allow the fluid to pass through. In some embodiments, the removal of excess fluid is achieved by providing a fluid that evaporates after being dispensed from the dispensing orifice.

[0250] Aspects of the present invention include methods of manufacturing a 3D structure including one or more input materials. The 3D structure can be used to simulate the normal function of tissue in a subject that may become diseased or damaged.

[0251] As described above, any suitable divalent cation can be used in combination with the subject methods to cure a chemically crosslinkable input material, including but not limited to Cd 2+, Ba 2+ , Cu 2+ , Ca 2+ , Ni 2+ , Co 2+ or Mn 2+ . In a preferred embodiment, Ca 2+ is used as the divalent cation. In a preferred embodiment, the chemically crosslinkable input material is contacted with a solution containing Ca 2+ to form a solidified fiber structure. In some embodiments, the concentration of Ca 2+ in the sheath solution ranges from about 80 mM to about 140 mM, such as about 90, 100, 110, 120, or 130 mM.

[0252] 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.

[0253] Aspects of the present invention include methods of using software tools to deposit one or more input materials in a patterned manner to form a solidified structure layer, the solidified structure layer being formed as a multi-layer 3D tissue structure. In some embodiments, the multi-layer 3D tissue structure includes a plurality of mammalian cells. Advantageously, by modulating the components of the subject input material (e.g., mammalian cell type, cell density, matrix components, active agents), the subject methods can be used to create multi-layer 3D tissue structures having a precisely controlled composition at any particular location in three-dimensional space. Thus, the subject methods facilitate the generation of complex three-dimensional tissue structures.

[0254] All patents and patent publications mentioned herein are hereby incorporated by reference in their entirety.

[0255] Examples

[0256] The following examples are provided to give a complete disclosure and description to those of ordinary skill in the art of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations 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 atmospheric or near atmospheric.

[0257] Example 1. Bioprinted Fiber Structure

[0258] This example demonstrates the ability to produce the bioprinted fiber structures of the present disclosure, where the structures are suspended during one or more of their printing, patterning, and / or post-printing processing. Figure 9Adepicts a frame 902 (e.g., substantially similar to the frame 202 at Figure 2A ), the frame having columns 906 (e.g., substantially similar to the columns 206 at Figure 2A ). Also shown is a fibrous structure 908, wherein the fibrous structure is generated by printing fibers around continuous opposing columns both in the vertical and horizontal directions to generate a grid type structure as shown. Figure 9B is an image of the fibrous structure 910 after coating the entire structure while the bioprinted fibrous structure 908 is fully suspended and then removing it from the frame.

[0259] Example 2. Immersion printing

[0260] This example demonstrates that components for suspending bioprinted fibrous structures as disclosed herein can be used to print bioprinted fibrous structures, wherein the components are immersed in a crosslinking agent bath during printing. Figure 10A Shows a bioprinted fibrous structure printed into a crosslinking agent bath using a suspending component as disclosed herein and then coated. Figure 10B Shows Figure 10A an enlarged view of a portion of the depicted structure. Figure 10C Shows a close-up view of the fibrous structure printed into the crosslinking agent bath, while Figure 10D shows a close-up view of the fibrous structure not printed into the crosslinking agent bath and dip-coated after printing. This example illustrates that a device for printing fibrous structures in a crosslinking agent bath using a suspending component (e.g., a frame as shown at Figures 2A to 2D ) has improved fidelity in terms of less shape deformation compared to a device that does not print the fibrous structure into the crosslinking agent bath.

[0261] Example 3. Print optimization and viability study of mini-devices

[0262] This example includes test conditions for a 4-layer fibrous structure of 10x10 mm, compared to control conditions for a 2-layer fibrous structure of 18x18 mm. Figure 11A is a schematic diagram of the 10x10 mm fibrous structure. Figure 11B is an image of the coated 10x10 mm fibrous structure attached to the frame. Figures 11C to 11D depicts the coated 10x10 mm fibrous structure separated from the frame.

[0263] The fibrous structure is coated with 0.5% SLG100 (alginate). The cell dose for the structure is 3K IEQ HepG2 aggregates. Live / dead staining is evaluated at 0 days ([[]] Figure 12A ) and 5 days ([[]] Figure 12B ) after printing.

[0264] This example also includes tests on a mini-device (10x10 mm, 4-layer device coated fiber structure) with a coating of 0.5% SLG100 and a core of 1.5% SLG100 with HA or just 1.5% SLG100. For each case, the cell dose was 3K IEQ HepG2 aggregates or primary rat islets (PRI). Figures 13A to 13B Images of the 10x10 mm fiber structure are shown on the frame ( Figure 13A ) and after coating separated from the frame ( Figure 13B ). Figure 14 Stability data are summarized at Figure 15A is an image of Structure 1 (with HA core), Figure 15B is an image of Structure 2 (with HA core), Figure 15C is an image of Structure 3 (with HA core), Figure 15D is an image of Structure 1 (conventional core), Figure 15E is an image of Structure 2 (conventional core), and Figure 15F is an image of Structure 3 (conventional core).

[0265] This example also includes viability and functionality tests on a coated 10x10 mm fiber structure loaded with PRI (coating 0.5% SLG100, cell dose: 3K IEQ PRI). Live / dead staining was evaluated at 0 days ([[]] Figure 16A ) and 3 days ([[]] Figure 16B ) after printing.

[0266] Example 4. Frame and Mesh Device Stability Test

[0267] This example demonstrates that devices printed using the frame of the present disclosure have enhanced stability compared to devices printed instead of the frame (i.e., printed onto the mesh without using the frame).

[0268] Experimental Design

[0269] In this example, the coated fiber structures tested were 18x18 mm and 2 layers thick. The coated fiber structures 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., crosslinker solution) had a flow rate of 55 μL / min when dispensed from the print head. Three structures were printed using the frame as disclosed herein, while three other structures were printed onto the mesh instead of the frame. The conformal coating was added to the bioprinted fiber structure when the bioprinted fiber structure was attached to the frame (devices printed relying on the frame), or the conformal coating was added when the bioprinted fiber structure was placed on the mesh (devices printed instead of the frame).

[0270] Stability test

[0271] The stability test was performed as follows. Each coated fibrous structure was cultured in 15 mL of PIMS medium in a 50 mL conical tube for 3 days. Each coated fibrous structure was subjected to orbital shaking at 125 rpm for 30 minutes or motor vehicle transportation for 30 minutes. Next, each coated fibrous structure was poured into a petri dish and washed three times with 10 mL of saline (aspirating the saline between each rinse). Each coated fibrous structure was lifted using a spatula and transferred between two petri dishes filled with saline to simulate device transfer at the surgical site. This was repeated 5 times. Finally, each coated fibrous structure was transferred to a moist plastic wrap, and each device was moved from one side of the membrane to the other three times using a small rod (to simulate repositioning of the device on the omentum).

[0272] Results

[0273] All three coated fibrous structures that relied on framework printing and coating as disclosed herein passed the stability test ( Figure 17 , and Figures 18A to 18C in contrast to Figures 18D to 18F ). All three fibrous grid structures printed onto the mesh instead of the framework did not pass the stability test, and microscopic images ( Figures 19A to 19C ) revealed that the fibers in the first layer leaked out of the coating (compare the images with Figures 19B to 19C and Figure 19A ). Figure 20 Images of other fibrous structures printed onto the mesh (but not coated) are depicted, showing a decrease in stability. Figure 20 The structure of

[0274] Example 5. The bioprinted cell therapy platform normalizes blood glucose control in diabetic rats

[0275] We developed a microfluidic bioprinting technology that combines biocompatible materials with clinically relevant cells to fabricate implantable tissues for therapeutic applications. Islet cell therapy has been clinically validated for type 1 diabetes (T1D), but relies on lifelong immunosuppression and is limited by the supply of cadaveric donor islets. We are developing a bioprinted pancreatic tissue therapy that can deliver allogeneic islets or stem cell-derived pancreatic β cells to T1D patients without the need for immunosuppression, which is achieved by encapsulation in a material that supports physiological function and protects these cells from direct attack by host immune cells.

[0276] This example demonstrates a bioprocess for encapsulating primary islets into bioprinted tissue implants for in vitro testing and in vivo functional studies. We evaluated the ability of bioprinted human islet tissue (xenograft) to restore glycemic control in diabetic, immunodeficient mice and adapted the process to deliver bioprinted primary rat islet tissue (allograft) to the omentum of diabetic rats. Finally, we developed a process to scale up this bioprinted pancreatic tissue manufacturing process to deliver implants to large animals and humans.

[0277] Materials and Methods

[0278] The main surgery performed on the animals was omental device implantation, as outlined in the text. The surgery was performed after treatment with STZ.

[0279] A. Prepare the animals for surgery.

[0280] The general concepts of "Rodent Anesthesia" (SOP ACC-01-2017), "Analgesia for Adult Mice and Rats Meloxicam SOP" (TECH 19), and "Local Anesthesia / Analgesia in Adult Mice and Rats Bupivacaine SOP" (TECH 16) were followed. The surgeries are explained below:

[0281] A1. Place the animal in an induction chamber on a heating pad (temperature should be approximately 38 °C) and induce anesthesia with isoflurane. Flush the chamber and move the animal to the maintenance circuit on a nose cone and heat support and maintain isoflurane anesthesia.

[0282] A2. Administer a small drop of lubricating eye gel in each eye. Place the animal in the prone position and administer supportive care fluid in the form of 0.9% saline or lactated Ringer's solution (LRS) subcutaneously at 20 mL / kg. Use a 25G, with various syringe sizes depending on the dose.

[0283] A3. Administer meloxicam subcutaneously at 1 mg / kg.

[0284] A4. Administer buprenorphine subcutaneously at 0.05 mg / kg.

[0285] A5. Place a paper towel under the animal to catch all the shaved hair when flipping it. Shave the abdominal skin of the animal with a clipper.

[0286] A6. Take a dry gauze and remove all the hair from and around the animal while pulling out the paper towel.

[0287] A7. Once the hair is cleared, take out a piece of gauze or cotton swab soaked in soap to clean the shaving area. The gauze needs to be damp but not dripping, and wrap the gauze around the finger. Clean the shaving area outward in a circular motion, and bubbles should be visible. Leave the soap on the animal for about 30 seconds, then wipe it with alcohol.

[0288] A8. Wipe the shaving area with a gauze or cotton swab containing 70% alcohol in an outward circular motion. Wipe again with a new gauze or cotton swab dipped in soap, but this time wipe off the oil on the skin. Let it stand for 30 seconds, then proceed to the next step.

[0289] A9. Clean the area with a new gauze or cotton swab soaked in alcohol.

[0290] A10. To inject a local anesthetic as a line block at the planned incision site. Lift the skin and insert the needle subcutaneously under the skin. When you inject, pull out the needle when a "blister" forms.

[0291] A110. Perform an additional skin preparation (soaked in soap and again with a gauze soaked in alcohol). Pinch the animal's toes to ensure it is properly anesthetized. Change gloves and wash hands with soap.

[0292] A12. During the entire surgery, monitor the color of the limbs (should be pink), respiratory rate and depth, and toe pinch every 5 minutes.

[0293] B. Prepare surgical instruments

[0294] B1. Put on clean examination gloves. This surgical procedure is performed using aseptic tip technique, and the gloved hand cannot touch any surface that must remain sterile.

[0295] B2. Open the sterile pack on the table, and aseptically take out the half-folded sterile area / drape, unfold it, so that the inside (sterile area) faces up when placing it near the surgical table.

[0296] B3. Take out a pair of sterile forceps to transfer all the instruments and supplies in the pack to the sterile area drape. Only the sterile parts of the instruments can enter the sterile area.

[0297] B4. Take out the "Glad Press and Seal Wrap", throw away the first 6", and pull out a piece to place on the animal. Avoid using the edges as they will be contaminated. Make sure not to touch the top surface with your fingers.

[0298] B5. Use a pair of forceps to pick up the cotton pad to 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, and grasp the needle with the forceps to cut a hole at the planned incision site. Do not allow any non-sterile items to contact the top surface of the drape.

[0299] B6. Use forceps to pinch the toes of the animal to ensure it is at the surgical anesthesia plane.

[0300] C. Surgery.

[0301] Follow the general concepts of "Rodent Survival Surgery" (SOP ACC-02-2017). All surgeries for immunodeficient rats are performed inside a biosafety cabinet in a laminar flow clean air workstation. The following explains the surgery:

[0302] C1. Use a toothed forceps to pick up the skin. Use a surgical blade to form a 20-mm incision along the midline of the skin approximately 2 cm below the xiphoid process.

[0303] C2. Once the skin incision is completed, grasp and lift the muscle with forceps, and first use a surgical blade to form a stab opening, then use scissors to form a 20-mm incision.

[0304] C3. Use a self-retaining tissue retractor to keep the peritoneal cavity incision open.

[0305] C4. Place a sterile gauze aseptically on the abdominal skin at the tail of the opening to prevent direct contact between the omentum and the skin.

[0306] C5. Locate the greater omentum and gently pull the omentum out of the opening using a pair of tissue forceps.

[0307] C6. Place the bioprinted implant aseptically in the middle of the exposed omentum.

[0308] Note: The bioprinted implant is composed of a non-reactive, non-rigid polymer that has no known biocompatibility issues. Each device is 20 x 20 x 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 under standard cell culture conditions (medium, 37°C / 5% CO2) for no more than four days before implantation. Shortly before implantation, wash the device with a sterile isotonic solution (such as saline or Ringer's buffer) to remove all traces of the medium.

[0309] C7. Fold the free end of the omentum over the implant and suture both sides of the omentum using a fine non-absorbable monofilament (e.g., 6-0 nylon or Prolene) to form a closed pouch.

[0310] C8. Gently slide the omentum pouch back into the abdominal cavity and close the incision with continuous or subcuticular sutures for the muscle layer and skin layer respectively.

[0311] C9. Moisten a 5-0 absorbable suture with saline. Use 1 suture pack per rat. Hold the needle vertically with a needle driver. Ensure that the suture does not touch the non-sterile areas of the needle holder or forceps.

[0312] C10. Insert the needle into one side of the muscle and withdraw it from the other side of the muscle. When withdrawing the needle, follow the direction of the needle. Tie a square knot and repeat this knot a total of 3 times. Leave approximately 2 - 3 mm of suture and cut the ends. Repeat until the muscle area is closed.

[0313] C11. Perform a subcuticular closure of the skin. On one side of the skin, insert the needle just beneath the skin layer and withdraw it from the farther side of the skin (deeper but still beneath the skin layer). Insert the needle on the opposite side of the skin, this time from deep to shallow. Ensure that the needle does not come out of the top of the skin. Tie three square knots.

[0314] C12. Once the skin layer is closed, remove the 25G needle and immerse it in Gluture. Remove the needle covered with Gluture to place it on top of the sutured skin. Use a pair of forceps to pinch the skin around the needle and slowly remove the needle. The sutured area is now properly closed.

[0315] Recovery of the rats:

[0316] C13. Turn off the isoflurane and supply oxygen to the rats via the nasal cone.

[0317] C14. Gently remove the drape and clean any blood from the rats.

[0318] C15. Once the rats regain their righting reflex, place the rats in a warmed recovery cage (cover the bare bottom of the cage with paper towels).

[0319] C16. Monitor the rats in the recovery cage until fully recovered from anesthesia and able to maintain their body temperature without supplemental heat (i.e., normal eating, drinking, walking, able to climb into the hut, able to groom).

[0320] C17. Once fully recovered, return the rats to the normal housing cage to stay with their cage mates.

[0321] D. Analgesia Plan

[0322] D1. Preoperative Analgesics: Before tissue incision, rats will receive a local anesthetic (bupivacaine 0.5 mg, 200 ul of 2.5 mg / ml solution) at the incision site, as well as injections of NSAID (meloxicam 1 mg / kg, SC) and buprenorphine (0.05 mg / kg, SC). Rats induced with liver disease will receive a combination of oral ibuprofen and low-dose buprenorphine to account for the reduced liver metabolism in these liver disease animals. According to TECH 09b for oral administration in rats (gavage), oral ibuprofen (30 mg / kg; ibuprofen liquid gel capsules were resuspended in water by vortexing, protected from light, and changed every 3 days) was administered at the time of surgery.

[0323] D2. Postoperative Analgesics: Day 1 and Day 2: Meloxicam (1 mg / kg, SC, SID) and buprenorphine (0.02 mg / kg, SC, BID)

[0324] These procedures follow TECH 16 (Local Anesthesia / Analgesia in Adult Mice and Rats - Bupivacaine SOP) and TECH 19 (Analgesia for Adult Mice and Rats - Meloxicam SOP). Rats induced with liver disease will receive a combination of oral ibuprofen and low-dose buprenorphine to account for the reduced liver metabolism in these liver disease animals. According to TECH 09b for oral administration in rats (gavage), oral ibuprofen (30 mg / kg; ibuprofen liquid gel capsules were resuspended in water by vortexing, protected from light, and changed every 3 days) was administered 6 hours after surgery. Overnight, animals continued to receive ibuprofen (1 mg / mL) in their drinking water. At 24 hours and 48 hours after surgery, animals received subcutaneous injections of buprenorphine (0.05 mg / kg).

[0325] E. Prophylactic Antibiotic Administration

[0326] Bio-printed implants are prepared using sterile materials and reagents and under sterile conditions. All surgeries are also performed in a sterile manner. However, to mitigate any potential infection risk, prophylactic antibiotics were added to the drinking water of rats from 3 days before surgery until 3 days after surgery (100 μg / mL enrofloxacin in the drinking water). Enrofloxacin is mainly excreted through the kidneys and is also suitable for rats with liver disease.

[0327] Other Procedures

[0328] F. Blood Sampling

[0329] Blood samples were collected from the rats weekly until the end of the study. Blood was collected from the lateral saphenous vein using TECH 02 SOP “Blood collection from the lateral saphenous vein in mice and rats”. The maximum blood sample volume per week was 150 ul, which was within the allowable limit for serial blood sampling as defined in “UBC ANIMAL CARE COMMITTEE POLICY 006, Policy on Acceptable Methods of Rodent Blood Withdrawal”

[0330] G. Streptozotocin administration was performed to induce diabetes

[0331] Streptozotocin (STZ) destroys the insulin-secreting cells of the pancreas, thus inducing diabetes. Since the use of STZ requires special handling precautions, all users and animal care technicians were made aware of the risks by the presence of SDS in the experimental area. For 1 week after STZ injection, cages containing animals treated with STZ were labeled accordingly

[0332] STZ supplied as a powder was reconstituted to a concentration of 30 mg / mL in acetate or citrate buffer (pH 4.5) shortly before injection

[0333] G1. To induce a type 1 diabetes (insulin-deficient) model, rats were injected intraperitoneally with STZ at a dose of 60 mg / kg in acetate or citrate buffer (for a standard 250 g rat, the maximum volume was approximately 0.5 mL). The IP injection procedure was performed following TECH 10b (Intraperitoneal Injection in the Adult Rat)

[0334] G2. After injection, the blood glucose levels of the animals were monitored daily. Those animals showing persistent hyperglycemia (blood glucose > 20 mmol / l for two consecutive readings) were used for the experiments

[0335] There is a risk of severe hypoglycemia within the first 24 - 48 hours. The initial cytotoxic destruction of the beta islet cells leads to an excessive release of insulin into the blood. To prevent any fatal hypoglycemia, sucrose water was provided during the induction period to reduce morbidity and mortality. For this purpose, 10% sucrose was added to the drinking water within 48 hours after STZ injection

[0336] H. Blood glucose measurement

[0337] Blood glucose is measured by placing a drop of blood (≤10 μL) on a blood glucose meter test strip (Lifescan Canada or equivalent).

[0338] H1. Obtain a small drop of blood from the tail vein using TECH 13 (UBC ACC tail prick SOP - rats) and apply it to the test strip.

[0339] H2. Apply gentle pressure to the tip of the tail with a 2x2 gauze for approximately 10 seconds to stop the blood flow.

[0340] I. Oral glucose tolerance test

[0341] The main function of the islets is to secrete insulin in response to elevated blood glucose levels. To monitor the in - vivo function of implanted islets, as described below, we stimulate the islets with an oral glucose dose after a short fasting period:

[0342] I1. Fast the animals for 4 hours (e.g., from 8 am to 12 pm).

[0343] I2. Obtain a blood sample (fasting, volume: 75 μL)

[0344] I3. Administer a glucose solution (3 g / kg, 150 μL) to the animals by oral gavage using a 1 mL syringe and a 20 - gauge and 38 - mm long flexible feeding tube (TECH 09 - Oral dosing in Mice and Rats will be followed)

[0345] I4. Obtain a blood sample again 30 minutes later (after glucose administration, volume: 75 μL).

[0346] J. Immunofluorescence protocol for insulin and CD31

[0347] Materials:

[0348] Insulin (C27C9) rabbit mAb (New England Biolabs), CD31 (PECAM - 1) mouse mAb (New England Biolabs), anti - rabbit IgG, Alexa 647 conjugate (New England Biolabs), anti - mouse IgG, Alexa 488 conjugate (New England Biolabs).

[0349] Procedure:

[0350] 1. Place the slides in a 60 °C oven for 15 minutes to start the dewaxing process

[0351] 2. Place the slide in a xylene-resistant holder

[0352] 3. Wash in xylene for 5 minutes (3X)

[0353] 4. Wash in 100% EtOH for 5 minutes (2X)

[0354] 5. Wash in 95% EtOH for 5 minutes

[0355] 6. Wash in 80% EtOH for 5 minutes

[0356] 7. Wash in 70% EtOH for 5 minutes

[0357] 8. Wash on a shaker in PBS for 5 minutes

[0358] 9. Place the slide in a beaker and cover with antigen retrieval buffer (10 mM citrate buffer, pH 6.0)

[0359] 10. Heat in a microwave at high power for 5 minutes (2X), ensuring the slide is always covered with buffer

[0360] 11. Wear heat-resistant gloves, remove the beaker from the microwave and place in a sink

[0361] 12. Cool the beaker under a gentle stream of cold running tap water for 5 - 10 minutes (do not let water hit the slide directly)

[0362] 13. Wash the slide in ddH2O for 5 minutes

[0363] 14. Wash the slide on a shaker in PBS for 5 minutes

[0364] 15. Remove excess water from the slide and circle the sample with a hydrophobic pen (Super Pap pen)

[0365] 16. Block the slide in a humid chamber in 5% BSA-PBS with 5% goat serum at room temperature for 1 hour

[0366] 17. Incubate overnight at 4°C in a humid chamber with primary antibody (diluted in 5% BSA-PBS with 5% goat serum):

[0367] a. Insulin: 1 / 500 dilution

[0368] b. CD31: 1 / 500 concentration

[0369] Day 3:

[0370] 1. Wash the slide in PBS for 10 minutes (3X)

[0371] 2. Incubate for 1 hour at room temperature in a dark humidity chamber with secondary antibodies (diluted in 5% BSA-PBS or PBS):

[0372] a. Anti-mouse: 1 / 1000 dilution

[0373] b. Anti-rabbit: 1 / 1000 dilution

[0374] 3. From this step onwards, the slides must be protected from light

[0375] 4. Wash the slides in PBS for 10 minutes (3X)

[0376] 5. Mount the slides with Fluoroshield with DAPI and seal the coverslip with clear nail polish

[0377] 6. Dry for 24 hours before imaging.

[0378] Islet Processing and In Vitro Bioprinting

[0379] Figure 21A A schematic diagram and bright-field image of bioprinted primary human islet tissue are depicted. Figure 21B Live / dead staining of bioprinted primary islets after 7 days in culture is shown (top: native islets; bottom: reaggregated islets). Figure 21C Data from glucose-stimulated insulin secretion (GSIS) assays using primary human (n = 8) and rat (n = 10) islets are depicted; mean + / − SEM.

[0380] Function of Bioprinted Pancreatic Tissue in Streptozotocin-Induced Rodent Diabetic Models

[0381] Immunodeficient (NSG) mice: IP implants

[0382] Figure 22A Random-fed blood glucose measurements within 80 days after streptozotocin (STZ) treatment and intraperitoneal (IP) implantation of bioprinted human islet tissue in NSG (NOD scidγ) mice (n = 5) are shown; day 0 represents the implantation time. Figure 22B Human C-peptide levels measured in mouse plasma using ELISA within 80 days are shown. Figure 22C Data from an oral glucose tolerance test (OGTT) performed on day 80 in NSG mice with bioprinted islet tissue or healthy untreated control mice are shown to evaluate the kinetics of blood glucose normalization after a fasting period and subsequent glucose challenge.

[0383] Immunodeficient (nude) rats: Omental implants

[0384] Figure 23A Shows blood glucose measurement results within 180 days after omental pouch implantation of bioprinted rat islet tissue in streptozotocin (STZ)-treated nude rats (n = 2). Figure 23B Shows hematoxylin and eosin (H&E, high and low magnification) and insulin (islets) or CD31 (endothelial cells) immunohistochemistry (IHC) performed on sections of fixed bioprinted tissue explanted at 180 days.

[0385] Immunocompetent (Sprague-Dawley) rats: Omental implantation

[0386] Figure 24A Shows blood glucose measurement results within 90 days after omental pouch implantation of bioprinted Lewis rat islet tissue in streptozotocin (STZ)-treated Sprague-Dawley (SD) rats (n = 3); explants were retrieved at 30, 60, and 90 days after surgical implantation and returned to the hyperglycemic state. Figure 24B Shows H&E and insulin (islets) or CD31 (endothelial cells) IHC performed on sections of fixed bioprinted tissue explanted at 60 days.

[0387] Bioprinted pancreatic tissue expansion for large animals

[0388] Figure 25A Is a schematic diagram showing that the biomanufacturing process involves tissue design in proprietary software and QC of bioprinted tissue, including confirmation of micro- and macroarchitecture, cell viability, and islet distribution. Figure 25B Shows a comparison of bioprinted pancreatic tissue for studies in rats and scaled-up tissue for large animals. Figure 25C Shows the viability of bioprinted neonatal pig islets confirmed up to 14 days after printing. GSIS demonstrated that the function of the bioprinted tissue was dose-compatible with human islets.

[0389] Conclusions

[0390] We developed a process (see Figure 26 ) to fabricate implantable tissue containing bioprocessed islets in a material that protects these allogeneic cells from host immune cell attack. This example shows that bioprinted pancreatic tissue: 1) maintains islet viability and function in vitro, 2) restores glycemic control in diabetic mouse and rat models, 3) supports islet function and immune protection for more than 90 days in a diabetic rat model, and 4) can be scaled up for large animal studies and ultimately delivered to T1D patients.

[0391] Example 6. Double dip coating of bioprinted fibrous structures

[0392] In this example, the coated fibrous structure being tested was 16x16 mm and two layers thick, and was printed on the device of the present disclosure including the vessels described herein. The coated fibrous structure includes a core (1.5% SLG100, with cells or dyes), 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 conformal coating and the outer conformal coating were added to the bioprinted fibrous structure while the bioprinted fibrous structure was attached to the frame (a device that relies on the frame for printing).

[0393] After printing a two-layer, 16x16 mm fibrous structure, the structure was crosslinked with 95% / 5% Ca / Ba in pH-buffered dH2O containing 15% polyethylene glycol (PEG) for 3 minutes. The crosslinking bath was removed by vacuum, and the fibrous structure was rinsed with TSC saline in the buffer channels on the print head. Then all solutions were removed by vacuum, and the fibrous structure was raised 3 to 5 mm from the receiving surface.

[0394] Then 1 mL of a first conformal coating solution of 2% SLG100 was pipetted into the vessel to uniformly coat the entire fibrous structure, ensuring that all fibers were covered with the solution starting from both the top and bottom. The fibrous structure was incubated in the first conformal coating solution for 10 seconds, after which the excess solution was removed from the vessel by vacuum.

[0395] Then 1 mL of a second conformal coating solution of Zwit-20 alginate was pipetted into the vessel to uniformly coat the entire fibrous structure, ensuring that all fibers were covered with the solution starting from both the top and bottom. The fibrous structure was incubated in the second conformal coating solution for 35 seconds, after which the excess solution was removed from the vessel by vacuum.

[0396] Then the fibrous structure was transferred to a 95% / 5% Ca / Ba bath and allowed to crosslink for 3 minutes, after which the fibrous structure was rinsed with saline.

[0397] Photographs of the fibrous structure with the inner and outer conformal coatings were taken and are shown in Figure 27 . As Figure 27 depicted, the diameter of the entire vertical fiber with the two conformal coatings is 1.004 mm, the inner conformal coating is between 40.8 μm (left, midpoint) and 50 μm (right, midpoint), and the outer conformal coating is between 88.1 μm (left, midpoint) and 90 μm (right, midpoint).

[0398] The embodiments and examples described above are merely illustrative and not restrictive. Those skilled in the art will recognize or be able to ascertain many equivalents of specific compounds, materials, and procedures using only routine experimentation. All such equivalents are considered to be within the scope of the appended claims and are covered by the appended claims.

Claims

1. A manufacturing platform for supporting the fabrication of a bioprinted fibrous structure during printing, patterning, and / or processing, wherein the platform includes a frame that defines a void and includes a plurality of posts on opposite sides of the frame for securing and suspending at least one crosslinkable fiber within the frame to form the fibrous structure, wherein during a 3D bioprinting process, a continuous length of the at least one fiber is printed around at least two, three, four, five, six, seven, eight, nine, ten, or more of the posts.

2. The manufacturing platform of claim 1, wherein the posts are positioned inside the frame; preferably wherein the posts are positioned on frame protrusions that extend into the void.

3. The manufacturing platform of claim 2, wherein the posts are spaced apart evenly or unevenly around the frame.

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

5. The manufacturing platform of claim 1, wherein at least a portion of the crosslinkable fiber includes a biomaterial.

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

7. The manufacturing platform of claim 1, wherein the fibrous structure includes a grid.

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

9. The manufacturing platform of claim 1, wherein the frame further includes a fiber cutting groove positioned adjacent to the void to permit a cutting tool to cut a portion of the fibrous structure.

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

11. A member for suspending a bioprinted fibrous structure during printing, patterning, and / or processing, wherein the member for suspension includes a frame coupled to a mounting bracket of a bioprinting system and / or a receiving surface, the frame including a plurality of posts that surround the frame to secure a continuous length of at least one crosslinkable fiber to form the fibrous structure.

12. A bioprinting system, comprising: The manufacturing platform according to any one of claims 1 to 10, or the member 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 relative to the dispensing orifice in three-dimensional space, the positioning unit being operably coupled to the receiving surface or the at least one dispensing orifice; And A dispensing member for dispensing the at least one crosslinkable fiber from the at least one dispensing orifice.

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

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

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

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

17. The bioprinting system according to claim 13, wherein the dispensing member comprises at least one pump; optionally, wherein the at least one pump comprises a pump assembly, the pump assembly comprising a plurality of pumps positioned in a radial array on a mounting bracket.

18. The bioprinting system according to any one of claims 12 to 17, further comprising at least one print head, the at least one print head comprising a plurality of microfluidic printing channels to selectively provide a corresponding plurality of materials.

19. The bioprinting system according to any one of claims 12 to 18, further comprising a vacuum chuck and an integrated container disposed on the receiving surface, the integrated container being formed by a wall protruding from the top surface of the vacuum chuck and defining the perimeter of the container; preferably, wherein the wall is configured to be inserted into a mating groove at the bottom of the frame.

20. A method for bioprinting a fibrous structure, the method comprising: providing the system according to claim 12, and dispensing a continuous length of the crosslinkable fiber around a plurality of the posts on the frame of the manufacturing platform to generate the fibrous structure.

21. The method according to claim 20, further comprising: adding a conformal coating to the entire outer surface of the fibrous structure while the fiber remains attached to the frame.

22. The method according to claim 20 or claim 21, further comprising: transporting the fibrous structure from one location to another while the fibrous structure remains attached to the frame.

23. The method according to claim 20 or claim 21, further comprising: storing the fibrous structure while the fibrous structure remains attached to the frame.

24. A bioprinted fibrous structure made by the method according to claim 20, comprising a continuous length of the crosslinkable fiber comprising at least one biomaterial, wherein the crosslinkable fiber comprises a solid core and at least one outer shell layer surrounding the solid core, wherein the bioprinted fibrous structure comprises at least two layers of a grid / mesh formed by the continuous crosslinkable fiber.

25. The bioprinted fibrous structure according to claim 24, wherein the thickness of each layer is from about 0.050 to about 3 mm.

26. The bioprinted fiber structure according to claim 25, having 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%.

27. The bioprinted fiber structure according to claim 24, having a fiber-to-fiber distance between about 1000 and 2000 μm, such as between about 1400 and 1600 μm, or about 1500 μm.

28. The bioprinted fiber structure according to claim 24, wherein the solid core comprises the at least one biomaterial; optionally wherein the solid core is segmented along the length of the fiber.

29. The bioprinted fiber structure according to claim 24, further comprising at least one inner shell layer surrounding the solid core, the at least one inner shell layer comprising at least one biomaterial; optionally wherein the solid core and / or the at least one inner shell layer are segmented along the length of the fiber.

30. The bioprinted fiber structure according to any one of claims 24 to 29, further comprising at least one conformal coating.

31. The bioprinted fiber structure according to claim 30, comprising a single conformal coating.

32. The bioprinted fiber structure according to claim 31, wherein the solid core comprises about 0.75% to 1.5% alginate, the at least one outer shell layer comprises about 1.5% to 2.5% alginate, and the coating comprises about 0.2% to 0.75% alginate.

33. The bioprinted fiber structure according to claim 30, comprising an inner conformal coating and an outer conformal coating.

34. The bioprinted fiber structure according to any one of claims 24 to 33, wherein the at least one biomaterial comprises islet cells.

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